Substance-encapsulating vesicle and process for producing the same

ABSTRACT

Provided is a method for easily and efficiently producing encapsulated substance vesicles wherein a substance is encapsulated in the cavity of vesicles obtained by polymer self-assembly. Empty vesicles that have membranes comprising a first polymer that is a block copolymer with uncharged hydrophilic segments and a first kind of charged segments and a second polymer with a second kind of charged segments that carry a charge that is the opposite of said first kind of charged segments as well as spaces that are enclosed by said membranes are mixed in an aqueous medium with the substance that is to be encapsulated in the spaces.

CROSS-REFERENCES TO RELATED APPLICATIONS

The current application is a continuation of U.S. patent applicationSer. No. 13/699,238, filed Nov. 20, 2012, allowed, which is a 371National Phase Application of PCT/JP2011/061790, which applicationclaims foreign priority to JP 2010-117823 and JP 2010-117821, both ofthem filed May 21, 2010, the disclosures of which are herebyincorporated by reference in their entireties for all purposes.

TECHNICAL FIELD

The present invention relates to a vesicle having a cavity in which asubstance is encapsulated, and also to a process of producing the same.

More specifically, the invention relates to a process of producing avesicle encapsulating a substance therein (substance-encapsulatingvesicle) easily and efficiently, by making the substance carried andencapsulated in the cavity of a vacant vesicle formed via self-assemblyof polymers, and also to a novel substance-encapsulating vesicleprepared by the process.

BACKGROUND ART

It is known that a vesicle can be formed via self-assembly of polymersof which the primary structures have been controlled precisely. Such avesicle is applicable to various molecular designs, and can serve a newfunction beyond the properties of the original polymers. Accordingly,the vesicle is being considered for use as a carrier for a drug deliverysystem (DDS) or as a biomaterial or functional material.

Patent Document 1 (JP-H8-188541A, of which the inventors overlap withthe present inventors) discloses a drug carrier in the form of anelectrostatically-united polymeric micelle formed via self-assembly of ablock copolymer having an uncharged segment and a charged segment.

Non-Patent Document 1 (Schlaad H. et al., Macromolecules, 2003, 36 (5),1417-1420) discloses a vesicle formed via self-assembly of a first blockcopolymer having poly(1,2-butadiene) block and poly(cesium methacrylate)block with a second block copolymer having polystyrene block andpoly(l-methyl-4-vinylpyridinium iodide) block (referred to as“polymersome”).

Patent Document 2 (WO2006/118260A, of which the inventors overlap withthe present inventors) discloses a vesicle formed via self-assembly of afirst block copolymer having an uncharged hydrophilic segment and acationic segment (e.g., PEG-polycation) with a second block copolymerhaving an uncharged hydrophilic segment and an anionic segment (e.g.,PEG-polyanion).

Non-Patent Document 2 (Anraku Y. et al., J. Am. Chem. Soc., 2010, 132(5), 1631-1636, of which the authors overlap with the present inventors)discloses a vesicle formed via self-assembly of a block copolymer havingan uncharged hydrophilic segment and a charged segment (e.g.,PEG-polycation) and a copolymer charged oppositely to the chargedsegment of the block copolymer (e.g., polyanion).

It is contemplated that such vesicles formed via self-assembly ofpolymers as mentioned above can encapsulate and carry various substanceswithin their cavities for desired applications (for overview, see, e.g.,Non-Patent Document 3: H. Nyin et al. Soft Matter, 2006, 2, 940-949; andNon-Patent Document 4: “liposome: New Developments in Applications”,supervised by Kazunari AKIYOSHI et al., NTS Inc., 2005).

A typical process of producing a vesicle encapsulating a substancewithin its cavity (hereinafter also referred to as“substance-encapsulating vesicle”) includes mixing a substance to beencapsulated (hereinafter also referred to as “encapsulation-targetsubstance”) with membrane component polymers or a preformed polymermembrane to cause formation of a polymer vesicle via self-assemblysimultaneously with enclosure of the substance into the vesicle cavity(hereinafter also referred to as “simultaneous mixing method”). Examplesinclude: emulsion method (see, e.g., Non-Patent Document 5: F. Szoka, Jret al., Proc. Natl. Acad. Sci. USA, 1978 75 (9) 4194-4198); andinstillation method using organic solution of lipids (see, e.g.,Non-Patent Document 6: Batzri, S. et al., Biochim. Biophys Acta 1973,298, 1015-1019).

However, the simultaneous mixing method has a drawback that presence ofthe encapsulation-target substance may affect vesicle formation processvia self-assembly, thereby preventing formation of a vesicle or, even ifnot, enclosure of the substance into the vesicle cavity. Another probleminvolved in this method is that it often requires use of organic solventwhich is detrimental to membrane formation, rendering the processcomplicated and causing damage to the encapsulation-target substance dueto the organic solvent. This method has still another drawback that itis difficult to form vesicles having uniform particle size and structureunless carrying out an additional step, which is likely to render theprocess complicated. Thus, this method lacks versatality, and is notpractical as a means for producing various kinds ofsubstance-encapsulating vesicles.

On the other hand, as a general method of producing a particleencapsulating a substance, there is a method in which anencapsulation-target substance is introduced into the cavity of anexisting vacant particle such that the substance is enclosed and carriedby the particle (hereinafter also referred to as “post-carrying method”)(see, e.g., Non-Patent Document 7: W. Tong et al. J. Phys. Chem. B,2005, 109, 13159-13165). This method could be an option for producingsubstance-encapsulating vesicles.

However, application of the post-carrying method to vesicles wouldrequire any additional means to introduce an encapsulation-targetsubstance beyond the membrane of a vacant vesicle into the vesiclecavity. A conceivable method includes: making the vacant vesicle swellto relax the membrane; penetrating the encapsulation-target substanceinto the cavity through cleavage which has occurred on the relaxedmembrane; and contracting the membrane to prevent release of theencapsulation-target substance. Another conceivable method includes:opening pores on the membrane of the vacant vesicle; introducing theencapsulation-target substance into the cavity through the pores; andclosing the pores to prevent release of the encapsulation-targetsubstance. However, these methods are cumbersome and complicated, toodisadvantageous to be put into practical use. In addition, the particlesize and the structure of the existing vacant vesicle would probably bedisturbed during the process of enclosure and carriage of theencapsulation-target substance. Accordingly, these methods have beenconsidered as being far from practical.

Another published method for lipid bilayer membrane vesicles such asliposomes includes integrating a channel protein into the lipid bilayermembrane (see, e.g., Non-Patent Document 8: Ranquin A, Versees W, MiereW, Steyaert J, Gelder PV., “Therapeutic Nanoreactors: CombiningChemistry and Biology in a Novel Triblock Copolymer”, Drug DeliverySystem, Nano Lett., 2005, 5:2220-4). However, this method is notpractical either, since the process is cumbersome and complicated andlacks versatality.

Thus, there is a demand for a process of easily and efficientlyproducing a substance-encapsulating vesicle, in which a substance isencapsulated in the cavity of a vesicle formed via self-assembly ofpolymers.

With regard to vacant vesicles for encapsulating and carrying an activeingredient in the vesicle cavity, there is still room for improvement inthe stability of carriage. In addition, it is still difficult to maketwo or more active ingredients carried by such a vesicle.

Thus, there is a demand for developing a new vesicle which can carry anactive ingredient with improved stability, and can also carry two ormore ingredients with improved controllability.

PRIOR ART REFERENCES Patent Documents

-   Patent Document 1: JP-H8-188541A-   Patent Document 2: WO2006/118260A

Non-Patent Documents

-   Non-Patent Document 1: Schlaad H. et al., Macromolecules, 2003, 36    (5), 1417-1420-   Non-Patent Document 2: Anraku Y. et al., J. Am. Chem. Soc., 2010,    132 (5), 1631-1636-   Non-Patent Document 3: H. Nyin et al. Soft Matter, 2006, 2, 940-949-   Non-Patent Document 4: “liposome: New Developments in Applications”,    supervised by Kazunari AKIYOSHI et al., NTS Inc., 2005-   Non-Patent Document 5: F. Szoka, Jr. et al., Proc. Natl. Acad. Sci.    USA, 1978 75 (9) 4194-4198-   Non-Patent Document 6: Batzri, S. et al., Biochim. Biophys Acta    1973, 298, 1015-1019-   Non-Patent Document 7: W. Tong et al., J. Phys. Chem. B, 2005, 109,    13159-13165-   Non-Patent Document 8: Ranquin A et al., Nano Lett. 2005; 5:2220-4

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

A problem to be addressed by the present invention is to provide aprocess of producing a substance-encapsulating vesicle easily andefficiently, in which vesicle a substance is encapsulated in the cavityof a vesicle formed via self-assembly of polymers.

Another problem to be addressed by the present invention is to provide anew vesicle which can carry an active ingredient with improvedstability, and can also carry two or more ingredients with improvedcontrollability.

Means to Solve the Problems

After intensive investigations in view of the aforementioned problems,the present inventors have finally found that a substance-encapsulatingvesicle having a substance encapsulated in the vesicle cavity can beproduced by preparing a vacant vesicle having a certain structure, andmixing the vacant vesicle with the encapsulation-target substance in anaqueous medium. This finding is very surprising, since although verysimple, this process enables efficient introduction of anencapsulation-target substance into a vacant vesicle using thepost-carrying method, without substantially impairing the structure ofthe vesicle.

In addition, in the course of developing the process, the presentinventors have found a novel vesicle of which the membrane contains anucleic acid.

Thus, an aspect of the present invention relates to a process ofproducing a substance-encapsulating vesicle, which encapsulates asubstance in a cavity thereof, comprising: providing a vacant vesiclehaving a membrane containing a first polymer, which is a block copolymerhaving an uncharged hydrophilic segment and a first charged segment, anda second polymer, which has a second charged segment charged oppositelyto the first charged segment, said membrane defining a cavity surroundedthereby; and mixing the vacant vesicle and the substance in an aqueousmedium.

Another aspect of the present invention relates to a vesicle comprising:a membrane containing a first polymer, which is a block copolymer havingan uncharged hydrophilic segment and a first charged segment, and asecond polymer, which has a second charged segment charged oppositely tothe first charged segment, said membrane defining a cavity surroundedthereby; and a substance encapsulated in the cavity.

Still another aspect of the present invention relates to a vesiclecomprising a membrane containing: a block copolymer having an unchargedhydrophilic segment and a cationic segment; and a nucleic acid; saidmembrane defining a cavity surrounded thereby.

Effects of the Invention

The present invention provides a method which can produces asubstance-encapsulating vesicle easily and efficiently, in which vesiclea substance is encapsulated in the cavity of a vesicle formed viaself-assembly of polymers. This method also provides a novelsubstance-encapsulating vesicle having high usefulness.

The present invention also provides a vesicle with a novel structure,having a cavity surrounded by a membrane containing a certain blockcopolymer and a nucleic acid.

BRIEF EXPLANATION OF THE DRAWINGS

FIGS. 1A and 1B are drawings for explaining the method of the presentinvention.

FIG. 2 is a drawing for explaining the structure of a vacant vesicle.

FIGS. 3A and 3B are drawings for explaining an embodiment of themembrane structure of a vacant vesicle.

FIGS. 4A and 4B are drawings for explaining another embodiment of themembrane structure of a vacant vesicle.

FIGS. 5A-5C are drawings for explaining an embodiment of the membranestructure of an intra-membrane nucleic acid-containing vesicle.

FIGS. 6A and 6B are graphs showing a relationship between the N+/P ratioand the mean particle diameter and the polydisperse index.

FIG. 7A is a transmission electron micrograph of a sample (N+/Pratio=1.2) in which an intra-membrane nucleic acid-containing vesiclewas formed, and FIG. 7B is a transmission electron micrograph of asample (N′/P ratio=2.0) in which no intra-membrane nucleicacidcontaining vesicle was formed.

FIGS. 8A-8D are the phase-contrast cryo-transmission electronmicrographs of a crosslinked intra-membrane nucleic acid-containingvesicle.

FIGS. 9A and 9B are graphs showing changes with time in the particlesize and shape of an intra-membrane nucleic acid-containing vesicle.

FIG. 10 is a graph showing the RNAi activity of an intra-membranenucleic acid-containing vesicle.

FIGS. 11A and 11B are drawings showing the intracellular uptake of siRNAby an intra-membrane nucleic acid-containing vesicle.

FIG. 12 is a graph showing substance encapsulation into anintra-membrane nucleic acid-containing vesicle.

FIG. 13 is a graph showing the particle distribution of thesubstance-encapsulating vesicle of Example V-1.

FIG. 14 is a graph showing the attenuation curve of thesubstance-encapsulating vesicle of Example V-1 measured by FCS.

FIG. 15 is a graph showing the particle distribution of thesubstance-encapsulating vesicle of Comparative Example V-1.

FIGS. 16A-16C are photofluorograms showing the result of evaluating thein vitro activity of the substance-encapsulating vesicle of ExampleVI-l.

FIG. 17 is a graph showing the particle distribution of thesubstance-encapsulating vesicle of Example VI-1.

FIG. 18 is a graph showing the attenuation curve of thesubstance-encapsulating vesicle of Example VI-1 measured by FCS.

FIG. 19 is a graph showing the particle distribution of thesubstance-encapsulating vesicle of Comparative Example VI-1.

FIG. 20 is a graph showing the attenuation curve of thesubstance-encapsulating vesicle of Example VII measured by FCS.

FIGS. 21A and 21B are the phase-contrast cryo-transmission electronmicrographs of the substance-encapsulating vesicle of Example VII.

FIG. 22 is a photofluorogram showing the result of evaluating the invitro activity of the substance-encapsulating vesicle of Example VIII.

FIGS. 23A and 23B are graphs showing changes in the structure of avacant vesicle by mixing.

MODE FOR CARRYING OUT THE INVENTION I: Definition

As used herein the term “vesicle” means a basic structure having amembrane of a unilamellar structure and a cavity (internal water phase)surrounded by the membrane.

As used herein the term “alkyl” represents a monovalent aliphaticsaturated hydrocarbon group. Unless otherwise specified, alkyl may be alinear or cyclic form, or a form in which a linear and a cyclic form arebound. Linear alkyl may be a straight- or branched-chain. Cyclic alkylmay be monocyclic or polycyclic, and in the case of polycyclic alkyl, itmay be a linked or fused ring, or a spiro ring.

As used herein the term “alkoxy” as a name of a group or part thereofrepresents a group in which the above alkyl is bound to one valence armof a divalent oxygen atom.

As used herein the term “aryl” as a name of a group or part thereofrepresents a monovalent aromatic hydrocarbon group. Unless otherwisespecified, aryl may be monocyclic or polycyclic, and in the case ofpolycyclic alkyl, it may be a linked or fused ring or a spiro ring.

As used herein, the number of carbons of a group is expressed as in“C₁₋₁₂ alkyl.” “C₁₋₁₂ alkyl” means that the number of carbons of thealkyl is 1 to 12.

As used herein the term “halogen atom” means a fluorine atom, a chlorineatom, a bromine atom, or an iodine atom.

As used herein, that a certain group is “optionally substituted” meansthat one or more hydrogen atoms contained in the group may besubstituted with one or more substituents (in the case of two or moresubstituents, they may be the same or different). The maximum number ofsubstituents can be easily determined by a person skilled in the artaccording to the structure and the number of hydrogen atoms contained inthe group.

As used herein the “substituent” is selected, unless otherwisespecified, from the group consisting of a halogen atom, an aryl group, ahydroxy group, an amino group, a carboxyl group, a cyano group, a formylgroup, a dimethylacetal formyl group, a diethylacetal formyl group, aC₁₋₆ alkoxycarbonyl group, a C₂₋₇ acylamide group, a siloxy group, atri(C₁₋₆ alkyl)siloxy group (C₁₋₆ alkyl may be the same or different)and a sillylamino group.

As used herein the term “shear stress” means a stress component forwhich the direction of a normal line of a force acting surface isdifferent from the force acting direction. The description “mixing undershear stress” means mixing so that shear stress is acted on an object tobe mixed. When an external force is applied on a fluid (e.g., an aqueousmedium) containing an object, “shear stress” usually acts on the objectpresent in the fluid. Thus, when an external force is applied to mix asubject solution described below (a liquid containing a vesicle and anencapsulation-target substance in an aqueous medium), for example,“shear stress” acts on the vesicle contained therein, and thus suchmixing corresponds to “mixing under shear stress”.

As used herein the term “RNAi” means RNA interference.

As used herein the term “siRNA (small interfering RNA)” means a lowmolecular weight (usually 19-27 base pairs, preferably 21-23 base pairs)double-stranded RNA involved in RNAi.

Now the embodiments of the present invention will be explained below.The following embodiments are only illustrative examples and the presentinvention may be carried out in any form.

II: Method for Producing a Substance-Encapsulating Vesicle (II-1:Summary)

As described above, in the simultaneous mixing method, which is arepresentative method for producing a substance-encapsulating vesicle,in addition to being uncertain about the success of encapsulation, itwas difficult to secure the stability of an encapsulation-targetsubstance or the uniformity of particle size and structure, andfurthermore the process was complicated. Thus, the method had manyproblems for it to be used as a universal method for producing a varietyof substance-encapsulating vesicles. On the other hand, there can beconceived to use a later supporting method which is mainly used forvacant particles. In this case, however, since a substance is introducedinto a cavity of a vacant vesicle by transcending the membrane of thevesicle, the method requires complicated procedures such as membranerelaxation by swelling of the vacant vesicle, pore formation in themembrane of the vacant vesicle, and the embedding of channel proteinsinto lipid bilayers, rendering the process very complicated. Besides itis difficult to control the particle size and structure of the vesicle,the method was considered to be hardly practical.

In contrast, in the method of producing a substance-encapsulatingvesicle of the present invention (hereinafter referred to as “theproduction method of the present invention”), a vacant vesicle of apredetermined structure is provided, which is mixed with anencapsulation-target substance in an aqueous medium to produce asubstance-encapsulating vesicle having encapsulated a substance in acavity thereof. Despite being such a simple method, it can efficientlyintroduce an encapsulation-target substance into a vacant vesicle evenwith a later supporting method, and besides vesicle structure is hardlyimpaired. This is a surprising finding.

The outline of the production method of the present invention will beexplained below with reference to FIGS. 1A and 1B. As used herein, FIGS.1A and 1B is a schematic diagram, and the present invention is notlimited to FIGS. 1A and 1B in any way.

As shown in FIG. 1A, a vacant vesicle 1 with a predetermined structurehaving a cavity 1 b surrounded by a membrane 1 a is provided, which,together with an encapsulation-target substance 9, is mixed in anaqueous medium. Thus, as shown in FIG. 1B, the encapsulation-targetsubstance 9 is introduced into the cavity 1 b by transcending themembrane 1 a of the vacant vesicle 1, and thereby asubstance-encapsulating vesicle 1′ encapsulating theencapsulation-target substance 9 in the cavity 1 b can be obtained.

It is a highly surprising finding that for the vesicle 1 obtained bypolymer self-assembly, which requires a highly sophisticated control ofthe structure, such a simple method can introduce and encapsulate thesubstance 9 into the cavity 1 b of the membrane 1 a. The productionmethod of the present invention is based on such a finding.

Now, the production method of the present invention will be explained indetail below, but the details of the vacant vesicle and theencapsulation-target substance will be described in different sections,and here other conditions and procedures will be explained.

(II-2: Mixing of a Vacant Vesicle and an Encapsulation-Target Substance)

The production method of the present invention includes a step of mixinga vacant vesicle and an encapsulation-target substance in an aqueousmedium (the liquid, a subject to be mixed, containing the vacant vesicleand the encapsulation-target substance may hereinafter be referred to as“a subject solution to be mixed”).

The method of mixing may not be specifically limited, and may be carriedout by a method of applying an external force to an aqueous medium.Thus, a method in which a vacant vesicle and an encapsulation-targetsubstance are added into an aqueous medium and allowed to stand so as todisperse them spontaneously (hereinafter referred to as“standing/dispersion mixing”) is excluded. As an example of mixing byapplying an external force to an aqueous medium, there can be mentionedagitation, shaking, impaction etc.

As an exemplary method that employs agitation, there can be mentioned amethod of agitating by rotating a container containing a subjectsolution to be mixed with a vortex mixer etc., and a method of directlyagitating the solution with an agitation blade, and the like.

As an exemplary method that employs shaking, there can be mentioned amethod of shaking a container containing a subject solution to be mixedwith a shaker etc.

As an exemplary method that employs impaction, there can be mentioned amethod of applying vibration by ultrasonic irradiation and various otherimpacts to a subject solution to be mixed, and the like.

By the above mixing, a substance is encapsulated in the cavity of avesicle and thus a substance-encapsulating vesicle can be produced.

The reason why a substance-encapsulating vesicle can be formed by mixingis not clear, but by applying an external force to an aqueous medium,shear stress is acted on the vacant vesicle (thereby, mixing by anexternal force application to an aqueous medium may be rephrased asmixing under shear stress). With such shear stress, it is believed, thestructure of the vacant vesicle may be disturbed and decomposed toroughly uniform small aggregates, which self-assemble again to uniformlyregenerate the vesicle, and at the same time an encapsulation-targetsubstance present in the aqueous medium may be encapsulated into thevesicle during vesicle regeneration (such a mechanism can also beestimated since vesicle decomposition into small aggregates by mixingwas also confirmed in a reference experiment described below). Thisphenomenon can hardly occur for a vesicle at a normal state, andtherefore the production method of the present invention that employssuch a phenomenon can be considered extremely innovative.

Considering the above mechanism, though the mixing condition is notlimited, it is preferred to select an appropriate condition that permitsthe sufficient disturbance of the vacant vesicle structure in an aqueousmedium and permits, after disturbance, the regeneration of the vesiclestructure. Usually, mixing may be carried out to the extent that a forcemay be applied to the entire subject solution to be mixed, andpreferably mixing may be carried out to the extent that the entiresubject solution to be mixed becomes roughly uniform.

A specific condition for mixing may vary with the mixing method, and inthe case of agitating, it may be usually carried out at a rotating speedof 500 rpm or higher, preferably 1000 rpm or higher, and usually 10000rpm or lower, preferably 5000 rpm or lower. When the rotating speed istoo low, a uniform substance-encapsulating vesicle can be hardly formed,whereas when it is too high, a vesicle may be impaired and destroyed.

The agitating time with a vortex mixer may vary with the rotating speed,and may usually be 60 seconds or more, preferably 120 seconds or more,and usually 10 minutes or less, preferably 5 minutes or less. When theagitating time is too short, a uniform substance-encapsulating vesiclecan be hardly formed, whereas when it is too long, a vesicle may beimpaired and destroyed.

A specific condition for using another mixing method (agitation with anagitation blade, shaking with a shaker, impacting with ultrasonicirradiation etc.) may be adjusted as appropriate so that a force may beapplied to a subject solution to be mixed, said force having a similardegree of strength to that obtained when agitation with a vortex mixeris carried out at the above rotating speed and agitation time.

When the above mechanism is taken into account, it is preferred tosecure a certain period of time, after mixing, during which the subjectsolution to be mixed is allowed to stand and the vesicle is uniformlyregenerated. Such a standing time is not limited, but may be one minuteor more, preferably 3 minutes or more.

The fact that a substance was encapsulated into the cavity of a vesiclecan be confirmed by such methods as the detection of changes indiffusion coefficient by fluorescence correlation spectroscopy (FCS),separation by size exclusion chromatography, direct examination bytransmission electron microscope, etc. In measurement of diffusioncoefficient by fluorescence correlation spectroscopy, the unevendistribution of the encapsulation-target substance in the vesicle (thusthe obtainment of a substance-encapsulating vesicle) can be confirmed bymeasuring changes in diffusion coefficient of a fluorescent sample usinga fluorescent sample as the encapsulation-target substance.

(II-3: Other Conditions Related to Mixing)

Usually, a solution (a subject solution to be mixed) containing a vacantvesicle and an encapsulation-target substance in an aqueous medium maybe prepared and subjected to the above mixing.

The type of the aqueous solvent is not limited. Preferably it may bewater, but a solvent (such as physiological saline, an aqueous buffersolution, and a mixed solvent of water and a water-soluble organicsolvent etc.) in which another ingredient was mixed with water can alsobe used as long as it does not badly affect the structure of a vacantvesicle or does not prevent the introduction of an encapsulation-targetsubstance into the vesicle. As an aqueous buffer solution, a 10 mM HEPESbuffer etc. may be used.

While a subject solution to be mixed may be prepared by any procedure,it is preferred that, since a vacant vesicle is prepared in an aqueousmedium as described below, an encapsulation-target substance may beadded to a prepared vacant vesicle-containing solution, which is thensubjected to mixing. The encapsulation-target substance may be added asit is to the vacant vesicle-containing solution, or may be added in theform of a solution in an aqueous medium or of a suspension etc.

The respective concentration of a vacant vesicle and anencapsulation-target substance in the subject solution to be mixed maynot be specifically limited, and may be decided considering thestructure of the vacant vesicle, the type of the encapsulation-targetsubstance, the desired encapsulation ratio of the encapsulation-targetsubstance to the vacant vesicle, etc.

However, from the viewpoint of enhancing the encapsulation efficiency ofan encapsulation-target substance into the vacant vesicle, theconcentration of the vacant vesicle in an aqueous medium may usually be0.1 mg/ml or more, especially 1 mg/ml or more, and usually 100 mg/ml orless, especially 10 mg/ml or less. When the concentration of the vacantvesicle is too low, the substance-encapsulating vesicle may not beformed. Since the particle size of the substance-encapsulating vesicleobtained is believed to depend on the concentration of the vacantvesicle, the concentration of the vacant vesicle should be decidedaccording to the desired particle size of the substance-encapsulatingvesicle.

Also, the concentration of the encapsulation-target substance in theaqueous medium, which varies with the property of theencapsulation-target substance, may usually be 0.1 mg/ml or more,especially 1 mg/ml or more, and usually 100 mg/ml or less, especially 50mg/ml or less. When the concentration of the encapsulation-targetsubstance is too low, the substance-encapsulating vesicle may not beformed.

While the pH of a mixture solution may not be specifically limited, andmay be adjusted as appropriate considering the conditions such as thestructure of the vacant vesicle, the type of the encapsulation-targetsubstance, and the respective concentration of the vacant vesicle andthe encapsulation-target substance in the mixture solution, it maypreferably be 5 or higher, more preferably 6.5 or higher, and preferably9 or lower, more preferably 7.5 or lower. pH may be easily adjusted byusing a buffer solution as a solvent. To adjust the pH of the mixturesolution and use it may be advantageous in retaining the structure ofthe vacant vesicle and allowing the vacant vesicle to efficientlyencapsulate the encapsulation-target substance.

While the ionic strength of a mixture solution can be adjusted asappropriate as long as it does not destroy the structure of a vacantvesicle or inhibit the encapsulation of an encapsulation-targetsubstance into the vacant vesicle, it may preferably be 0 mM or more,more preferably 10 mM or more, and preferably 200 mM or less, morepreferably 50 mM or less.

While the temperature during mixing of a mixture solution may not belimited as long as it does not destroy the structure of a vacant vesicleor inhibit the encapsulation of an encapsulation-target substance intothe vacant vesicle, it may preferably be 10° C. or higher, morepreferably 20° C. or higher, and preferably 80° C. or lower, morepreferably 50° C. or lower.

After mixing, the formed substance-encapsulating vesicle may immediatelybe subjected to the desired use, or in order to equilibrate the system,time for allowing the mixed liquid to stand may be secured. While thetime for allowing the mixture solution to stand may vary depending onthe conditions such as the efficiency of forming asubstance-encapsulating vesicle, it may preferably be 50 hours or less,more preferably 30 hours or less. When a cross-linker may not be used asdescribed below, it may sometimes be preferred not to allow time longerthan is required for uniform regeneration of the vesicle, since the sizeof the formed substance-encapsulating vesicle may tend to increase withtime.

When a cross-linker is used, it may be added to a mixture solutioncontaining the formed substance-encapsulating vesicle, and thecross-linker may be added and mixed. While the cross-linker may be addedas it is, an aqueous solution containing the cross-linker may beprepared and this solution may be added. The conditions for preparing anaqueous solvent, pH, temperature, ionic strength etc. in the preparationof an aqueous solution of a cross-linker may be similar to thosedescribed above for the mixture solution.

III: A Vacant Vesicle (III-1: Structure of a Vacant Vesicle)

In the production method of the present invention, a vesicle comprisinga membrane containing a first polymer, which is a block copolymer havingan uncharged hydrophilic segment and a first charged segment, and asecond polymer, which has a second charged segment charged oppositely tothe first charged segment, said membrane defining a cavity surroundedthereby is used as a vacant vesicle.

An example of the structure of a vacant vesicle will be explained withreference to FIGS. 2, 3A-3B, and 4A-4B. Any of FIGS. 2, 3A-3B, and 4A-4Bare a schematic diagram, and the present invention is not limited tothese drawings in any way.

FIG. 2 is a partially broken view of a vesicle 1. As shown in FIG. 2,the vesicle 1 has a membrane 1 a and a cavity 1 b surrounded by themembrane 1 a.

FIG. 3A is a partially enlarged sectional view of the membrane 1 a ofthe vesicle 1 according to an embodiment of the present invention. Themembrane 1 a shown in FIG. 3A has a trilaminar structure comprising anouter layer 1 a _(o), an intermediate layer 1 a _(m), and an inner layer1 a _(i), and mainly formed by a first polymer 2 and a second polymer 3.

FIG. 3B is an enlarged view of the first polymer 2 and the secondpolymer 3 shown in FIG. 3A. As shown in FIG. 3B, the first polymer 2 isa block copolymer having an uncharged hydrophilic segment 2 a and afirst charged segment 2 b, and the second polymer 3 is a polymer thathas a second charged segment 3 charged oppositely to the first chargedsegment 2 b. Preferably, as shown in FIG. 3B, the uncharged hydrophilicsegment 2 a may form the outer layer 1 a _(o) of the membrane 1 a, andthe first charged segment 2 b and the second charged segment 3 may beelectrostatically bound to form the intermediate layer 1 a _(m). Andpreferably, the uncharged hydrophilic segment 2 a may mainly form theinner layer 1 a, of the membrane 1 a.

FIG. 4A is a partially enlarged sectional view of the membrane 1 a ofthe vesicle 1 according to an embodiment of the present invention. Themembrane 1 a shown in FIG. 4A also has a trilaminar structure comprisingthe outer layer 1 a _(o), the intermediate layer 1 a _(m), and the innerlayer 1 a _(i), and mainly formed by the first polymer 2 and a secondpolymer 3′.

FIG. 4B is an enlarged view of the first polymer 2 and the secondpolymer 3′ shown in FIG. 4A. As shown in FIG. 4B, the first polymer 2 isa block copolymer having the uncharged hydrophilic segment 2 a and thefirst charged segment 2 b, and the second polymer 3′ is a polymer thathas the uncharged hydrophilic segment 3 a and the second charged segment3 b charged oppositely to the first charged segment 2 b. Preferably, asshown in FIG. 4A, one or both of the uncharged hydrophilic segment 2 aand 3 a may form the outer layer 1 a _(o) of the membrane 1 a, and thefirst charged segment 2 b and the second charged segment 3 b may beelectrostatically bound to form the intermediate layer 1 a _(m). Andpreferably, one or both of the uncharged hydrophilic segments 2 a and 3a may form the inner layer 1 a _(i) of the membrane 1 a.

It is believed, but not intended to be bound by a theory, that themechanism in which the vesicle 1 is formed from the first polymer 2 andthe second polymer 3 or 3′ is as follows. Thus, the first polymer 2 andthe second polymer 3 or 3′ shown in FIG. 3B and FIG. 4B, when disposedin a system (for example, in an aqueous medium) that can generate theinteraction of electric charges, self-assemble, and, as shown in FIG. 3Aand FIG. 4A, the first charged segment 2 b and the second chargedsegments 3 and 3 b, charged oppositely to each another, areelectrostatically bound to form the intermediate layer 1 a _(m) andsimultaneously the uncharged hydrophilic segments 2 a and 3 a aredisposed outside thereof forming the outer layer 1 a _(o). Preferably,inside the intermediate layer 1 a _(i) as well, the unchargedhydrophilic segments 2 a and 3 a may be mainly disposed to form theinner layer 1 a _(i). Thus, it is believed, the membrane 1 a of atrilaminar structure shown in FIG. 3A and FIG. 4A may be formedresulting in the formation of the vesicle 1 shown in FIG. 2.

While the membrane 1 a of the vesicle 1 may only be formed of the firstpolymer 2 and the second polymer 3 or 3′, it may contain an additionalcomponent as long as the above structure may largely be maintained. Theadditional component may be not limited and may include, for example, across-linker, a charged polymer, a charged molecule, etc. Thecross-linker will be explained in detail below.

Also, as described below, since the vesicle 1 may usually be prepared inan aqueous medium, and since the inner layer 1 a _(i) of the membrane 1a may be mainly composed of the uncharged hydrophilic segments 2 a and 3a, an aqueous medium may usually be present in the cavity 1 b of thevesicle 1 (therefore, herein the cavity 1 b may sometimes be expressedas “internal water phase”). However, another substance may be present inthe cavity 1 b.

While the shape of the vesicle 1 may not be limited, it may usually bespherical or roughly spherical.

While the particle size of the vesicle 1 may vary with the type and themass ratio of the first polymer 2 and the second polymers 3 and 3′, thepresence or absence of a cross-linker, the environment (the type of theaqueous medium) surrounding the vesicle 1, etc., it may preferably be 10nm or more, more preferably 50 nm or more, and preferably 1000 nm orless, more preferably 400 nm or less, and even more preferably 200 nm orless.

While the thickness of the membrane 1 a of the vesicle 1 may vary withthe type and the mass ratio of the first polymer 2 and the secondpolymers 3 and 3′, the presence or absence of a cross-linker, theenvironment (the type of the aqueous medium) surrounding the vesicle 1,etc., it may preferably be 5 nm or more, more preferably 10 nm or more,and preferably 30 nm or less, more preferably 15 nm or less.

(III-2: First and Second Polymers)

A vacant vesicle for use in the production method of the presentinvention may have a membrane composed of a first polymer and a secondpolymer.

The first polymer is a block copolymer having an uncharged hydrophilicsegment and a first charged segment. The first polymer may be of onlyone type or two or more types that are used together in any combinationor any ratio.

The second polymer is a polymer having a second charged segment chargedoppositely to the first charged segment. It may be a polymer composedonly of the second charged segment, or may be a block copolymer havingan uncharged hydrophilic segment in addition to the second chargedsegment. The second polymer may be of only one type or two or more typesthat are used together in any combination or any ratio. When two or moretypes are used, a second polymer composed only of the second chargedsegment and a second polymer having an uncharged hydrophilic segment inaddition to the second charged segment may be used together.

The first polymer and the second polymer each may have another segmentin addition to the above-mentioned segment, respectively.

(III-2a: Uncharged Hydrophilic Segment)

The first polymer has an uncharged hydrophilic segment. The secondpolymer also has an uncharged hydrophilic segment.

The uncharged hydrophilic segment is a polymer segment having unchargedand hydrophilic properties. As used herein the term “uncharged”indicates that the segment as a whole is neutral. For example, a segmenthaving no positive or negative charge may be mentioned. Also, even ifthe segment has a positive or negative charge in the molecule, it stillcorresponds to “uncharged” when the local effective charge density isnot high and the electric charge of the segment as a whole has beenneutralized to the extent being unable to prevent vesicle formation byself-assembly. “Hydrophilicity” indicates that it exhibits solubility ina sequence medium.

The type of the uncharged hydrophilic segment may not be limited. It maybe a segment comprising repeating single units, or a segment containingrepeating units of two or more types in any combination or any ratio. Asa specific example of the uncharged hydrophilic segment, there can bementioned polyalkylene glycol, poly(2-oxazoline), polysaccharide,polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide,polymethacrylamide, polyacrylate, polymethacrylate,poly(2-methacryloiloxyethyl phosphorylcholine), a peptide or protein ora derivative thereof having an isoelectric point of about 7, and thelike. Among them, polyalkylene glycol, poly(2-oxazoline) etc. may bepreferred, with polyalkylene glycol being most preferred. Aspolyalkylene glycol, polyethylene glycol and polypropylene glycol may bementioned with polyethylene glycol being preferred.

While the molecular weight of the uncharged hydrophilic segment may notbe limited, it may preferably have a molecular weight within apredetermined range from the viewpoint of promoting the self-assembly ofa first polymer and a second polymer and efficiently producing a uniformvesicle. While the specific range of molecular weight may vary with thetype of the uncharged hydrophilic segment or combination with a chargedsegment, the molecular weight (Mw) may preferably be 500 or more, morepreferably 1000 or more, and preferably 15000 or less, more preferably5000 or less when polyethylene glycol is used as the unchargedhydrophilic segment. While the number of repeating units of theuncharged hydrophilic segment may not be limited, it may usually bedecided according to the type of the repeating units so that themolecular weight of the uncharged hydrophilic segment may satisfy theabove range of molecular weight.

By using an uncharged hydrophilic segment that satisfies the abovecondition, it becomes possible to prevent aggregation and precipitationof the first polymer and the second polymer in an aqueous solution,leading to stabilization of the polymers, and to efficiently construct avesicle.

(III-2b: Charged Segment)

The first charged segment contained in the first polymer and the secondcharged segment contained in the second polymer are charged segmentscharged oppositely to each other. Thus, when the first charged segmentis a cationic segment, then the second charged segment will be ananionic segment, and when the first charged segment is an anionicsegment, then the second charged segment will be a cationic segment.

(III-2b-1: Cationic Segment)

A cationic segment is a polymer segment that has a cationic group andthat exhibits a cationic property. However, the cationic segment mayhave a few anionic groups unless it prevents vesicle formation by theself-assembly of the first polymer and the second polymer.

The type of the cationic segment may not be limited. It may be a segmentcomprising repeating single units, or a segment containing repeatingunits of two or more types in any combination or any ratio. As acationic segment, polyamine etc may be preferred, and a polyamino acidor a derivative thereof having an amino group at the side chain thereofmay be specifically preferred. As a polyamino acid or a derivativethereof having an amino group at the side chain thereof,polyaspartamide, polyglutamide, polylysine, polyarginine, polyhistidine,and derivatives thereof may be mentioned, with a polyaspartamidederivative and polyglutamide derivative being most preferred.

While the molecular weight of a cationic segment may not be limited, itmay preferably have a molecular weight within a predetermined range fromthe viewpoint of promoting the self-assembly of a first polymer and asecond polymer and efficiently producing a uniform vesicle. While thenumber of repeating units of the cationic segment may not be limited, itmay usually be decided according to the type of the repeating units sothat the molecular weight of the cationic segment may satisfy the aboverange of molecular weight. Specifically when a polyaspartic acidderivative is used as a cationic segment, the number of repeating unitsthereof may preferably be 10 or more, more preferably 50 or more, andpreferably 200 or less, more preferably 100 or less.

By using a cationic segment that satisfies the above condition, itbecomes possible to prevent aggregation and precipitation of the firstpolymer and the second polymer in an aqueous solution leading to thestabilization of the polymers, and to efficiently construct a vesicle.

(III-2b-2: Anionic Segment)

An anionic segment is a polymer segment that has an anionic group andthat exhibits an anionic property. However, the anionic segment may havea few anionic groups unless it prevents vesicle formation by theself-assembly of the first polymer and the second polymer.

The type of the anionic segment may not be limited, either. It may be asegment comprising repeating single units, or a segment containingrepeating units of two or more types in any combination or any ratio. Asan anionic segment, polycarboxylic acid, polysulfonic acid, andpolyphosphoric acid (nucleic acid etc.) may be preferred, and apolyamino acid or a derivative thereof having a carboxyl group at theside chain thereof or a derivative thereof may be preferred with nucleicacid being specifically preferred.

As a polyamino acid or a derivative thereof having a carboxyl group atthe side chain thereof, polyaspartic acid, polyglutamic acid,polycarboxylic acid obtained by allowing an appropriate amount ofaconitic acid anhydride or citraconic acid anhydride to act on the aminogroup of a polyamino acid, which is the above polycation, or aderivative thereof having an amino group at the side chain, andderivatives thereof may be mentioned, with polyaspartic acid andpolyglutamic acid being most preferred.

As a nucleic acid, a single-stranded or double-stranded DNA or RNA maybe mentioned. A nucleic acid may be a functional nucleic acidappropriate for use in a vesicle. As a functional nucleic acid, siRNA,miRNA (micro RNA), antisense RNA, antisense DNA, ribozyme, DNA enzymeetc. may be mentioned. They may be selected according to the use of avesicle. For example, when a vesicle is used as a DDS for RNAi, siRNAmay be used as a nucleic acid. A nucleic acid may be modified. Asexamples of a modified nucleic acid, a nucleic acid to which ahydrophobic functional group such as cholesterol and vitamin E is boundmay be mentioned.

While the molecular weight of an anionic segment may not be limited, itmay preferably have a molecular weight within a predetermined range fromthe viewpoint of promoting the self-assembly of a first polymer and asecond polymer and efficiently producing a uniform vesicle. While thenumber of repeating units of the cationic segment may not be limited, itmay usually be decided according to the type of the repeating units sothat the molecular weight of the anionic segment may satisfy the aboverange of molecular weight. Specifically when a polycarboxylic acid,polysulfonic acid or nucleic acid is used as an anionic segment, thenumber of repeating units thereof may preferably be 10 or more, morepreferably 50 or more, and preferably 200 or less, more preferably 100or less.

By using an anionic segment that satisfies the above condition, itbecomes possible to prevent aggregation and precipitation of the firstpolymer and the second polymer in an aqueous solution leading tostabilization of the polymers, and to efficiently construct a vesicle.

(III-2c: Combination of an Uncharged Hydrophilic Segment and a ChargedSegment)

The combination of an uncharged hydrophilic segment and a first chargedsegment contained in the first polymer, and the combination of anuncharged hydrophilic segment and a second charged segment in a case inwhich the second polymer has an uncharged hydrophilic segment inaddition to a second charged segment are both not limited, and anyuncharged hydrophilic segment and any charged segment can be combined(in the following description, the first charged segment and the secondcharged segment may be collectively expressed as “charged segment”).

The number of the uncharged hydrophilic segments and that of the chargedsegments are also arbitrary, and may be one or more. In the case of twoor more, they may be the same or different.

While the binding form of an uncharged hydrophilic segment and a chargedsegment may not be limited, they may be directly bound or may be boundvia a linking group.

As an example of a linking group, there can be mentioned a hydrocarbongroup having a valency corresponding to the total number of theuncharged hydrophilic segments and the charged segments. The hydrocarbongroup as a linking group may be an aliphatic or an aromatic group, or agroup in which they are linked, and in the case of an aliphatic group,it may be saturated or unsaturated, and may be straight, branched orcircular. While the molecular weight of the hydrocarbon group as alinking group may not be specifically limited, it may usually be 5000 orless, preferably 1000 or less. As an example of a hydrocarbon group as alinking group, a gallic acid derivative, a 3,5-dihydroxy benzoic acidderivative, a glycerin derivative, a cyclohexane derivative, L-lysineetc. may be mentioned, with a 3,5-dihydroxy benzoic acid derivativebeing preferred.

As an example of a linking group, a disulfide group can be mentioned. Adisulfide group may be used to link one uncharged hydrophilic segmentand one charged segment. By linking an uncharged hydrophilic segment anda charged segment via a disulfide group, it becomes possible to cleavethe disulfide group by the environment surrounding the vesicle or anexternal action and to alter the form and properties of the vesicle. Byusing this, it is believed, when a drug is encapsulated in the vesicleand the substance-encapsulating vesicle is used as a DDS for drugdelivery, it becomes possible to cleave the disulfide group in vivo andthereby to promote the release of the substance encapsulated in thevesicle.

While the ratio of a first charged segment and a second charged segment(the ratio of a cationic segment and an anionic segment) and the ratioof an uncharged hydrophilic segment and a charged segment are alsoarbitrary, they may preferably be selected based on the followingcriteria, from the viewpoint of promoting the self-assembly of a firstpolymer and a second polymer and efficiently producing a uniformvesicle.

First, the ratio of a cationic segment and an anionic segment maypreferably be adjusted so that the C/A ratio defined in the followingequation (i) is usually 0.3, preferably 0.5 or more, more preferably 0.6or more, and usually less than 3.0, preferably 2.0 or less, morepreferably 1.7 or less.

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 1} \right\rbrack & \; \\{\begin{matrix}{C\text{/}A\mspace{14mu} {ratio}} \\\left( {{mole}\mspace{14mu} {ratio}} \right)\end{matrix} = \frac{\begin{bmatrix}{{moles}\mspace{14mu} {of}\mspace{14mu} {cationic}\mspace{14mu} {groups}} \\{{in}\mspace{14mu} {the}\mspace{14mu} {first}\mspace{14mu} {and}\mspace{14mu} {second}} \\{polymers}\end{bmatrix}}{\begin{bmatrix}{{moles}\mspace{14mu} {of}\mspace{14mu} {anionic}\mspace{14mu} {groups}} \\{{in}\mspace{14mu} {the}\mspace{14mu} {first}\mspace{14mu} {and}\mspace{14mu} {second}} \\{polymer}\end{bmatrix}}} & {{Equation}\mspace{14mu} (i)}\end{matrix}$

wherein, the moles of cationic groups and anionic groups in the firstand second polymers depend on the structure of the cationic segment andthe anionic segment, and can be determined by a common potentiometric(acid/base) titration.

The ratio of the uncharged hydrophilic segment and the charged segmentin the first and second polymers may preferably be decided consideringthe ratio of the cationic segment and the anionic segment that satisfiesthe above range of the C/A ratio. Specifically, the molecular weightratio X of the uncharged hydrophilic segment defined by the followingequation (ii) may preferably be kept in the range of usually 0.01 ormore, preferably 0.05 or more, and usually 0.35 or less, preferably 0.1or less.

In a case where one each of the cationic segment (assumed to have onepositive electric charge per monomer) and the anionic segment (assumedto have one negative electric charge per monomer) is used, and anuncharged hydrophilic segment is introduced into at least one of them(i.e., a case where the first polymer is a block copolymer having acationic or an anionic segment and an uncharged hydrophilic segment, andthe second polymer is a single polymer of an anionic or a cationicsegment or a block copolymer having an uncharged hydrophilic segment inaddition to it), its X is defined by the following equation:

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 2} \right\rbrack & \; \\{X = \frac{M_{NA} + {M_{NC}*\frac{\left( {C/A} \right)*P_{A}}{P_{C}}}}{M_{NA} + M_{A} + {\left( {M_{C} + M_{NC}} \right)*\frac{\left( {C/A} \right)*P_{A}}{P_{C}}}}} & {{Equation}\mspace{14mu} ({ii})}\end{matrix}$

M_(M) represents the molecular weight of the uncharged hydrophilicsegment linked to the anionic segment,

M_(NC) represents the molecular weight of the uncharged hydrophilicsegment linked to the cationic segment,

M_(C) represents the molecular weight of the cationic segment,

M_(A) represents the molecular weight of the anionic segment,

P_(C) represents the degree of polymerization of the cationic segment,and

P_(A) represents the degree of polymerization of the anionic segment.

(III-2d: Specific Examples of the First and Second Polymers)

As specific examples of the first and second polymers, the following[Example 1] and [Example 2] may be mentioned.

Example 1

The following (A1) is used as the first polymer and the following (B1)is used as the second polymer.

(A1) A block copolymer having an uncharged hydrophilic segment and ananionic segment.

(B1) A block copolymer of the following (i) and/or a polymer of thefollowing (ii):

(i) A block copolymer having an uncharged hydrophilic segment and acationic segment.

(ii) A polymer having a cationic segment (but, having no unchargedhydrophilic segment).

Example 2

The following (A2) is used as the first polymer and the following (B2)is used as the second polymer.

(A2) A block copolymer having an uncharged hydrophilic segment and ananionic segment.

(B2) A block copolymer of the following (III) and/or a polymer of thefollowing (iv):

(III) A block copolymer having an uncharged hydrophilic segment and ananionic segment.

(iv) A polymer having an anionic segment (but, having no unchargedhydrophilic segment).

As used herein, polymers having no uncharged hydrophilic segment, as inthe above (B1) (i) and (ii) and (B2) (iv) polymers, may be calledhomopolymers for convenience.

While cationic segments in each of the above (B1) (i) and (ii) and (A2)polymers may not be specifically limited, there may preferably bementioned, for example, those derived from polypeptides having acationic group at the side chain thereof.

Similarly, while anionic segments in each of the above (A1) and (B2)(III) and (iv) polymers may not be specifically limited, there maypreferably be mentioned, for example, those derived from polypeptides ornucleic acids having an anionic group at the side chain thereof.

More specifically, as each block copolymer of the above (A1) and (B2)(III), there can be preferably mentioned, for example, those representedby the following general formula (I) and/or (II):

wherein, in the structural formula of the general formulas (I) and (II),the segments having a number (degree of polymerization) of repeatingunits of “m” are uncharged hydrophilic segments derived from PEG(hereinafter referred to as “PEG segments”), and the segments thatcombine sections having a number of repeating units of “n-y” andsections having a number of repeating units of “y” are anionic segmentsderived from polyanions (hereinafter referred to as “polyanionicsegments”).

In the general formulas (I) and (II), R^(1a) and R^(1b) represent,independently from each other, a hydrogen atom or an unsubstituted orsubstituted straight or branched C₁₋₁₂ alkyl group. As a straight orbranched C₁₋₁₂, there can be mentioned methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl,undecyl and the like. As a substituent when they are substituted, therecan be mentioned an acetalized formyl group, a cyano group, a formylgroup, a carboxyl group, an amino group, a C₁₋₆ alkoxycarbonyl group, aC₂₋₇ acylamido group, the same or different tri-C₁₋₆ alkylsiloxy group,a siloxy group, and a sillylamino group. As used herein acetalizationmeans the formation of an acetal section by the reaction of formylcarbonyl with 2 molecules of alkanol having 1-6 carbons or an optionallybranched alkylenediol having 2-6 carbons, and thus is also a method forprotecting the carbonyl group. For example, when the substituent is anacetalized formyl group, it can be hydrolyzed under an acidic mildcondition to be converted to another substituent, a formyl group (—CHO)(or an aldehyde group).

In the general formulas (I) and (II), L¹ and L² represent a linkinggroup. Specifically, L¹ may preferably be —(CH₂)_(b)—NH— (b is aninteger of 1-5), and L₂ may preferably be —(CH₂)_(c)—CO— (c is aninteger of 1-5).

In the general formulas (I) and (II), R^(2a), R^(2b), R^(2c) and R^(2d)represent, independently from each other, a methylene group or anethylene group. When both of R^(2a) and R^(2b) are a methylene group,they correspond to a poly(aspartic acid derivative), and when they arean ethylene group, they correspond to a poly(glutamic acid derivative),and when both of R^(2c) and R^(2d) are a methylene group, theycorrespond to a poly(aspartic acid derivative), and when they are anethylene group, they correspond to a poly(glutamic acid derivative). Inthese general formulas, when R^(2a) and R^(2b) (R^(2b) and R^(2a))represent both of a methylene group and an ethylene group, and whenR^(2c) and R^(2d) (R^(2d) and R^(2c)) represent both of a methylenegroup and an ethylene group, repeating units of the aspartic acidderivative and the glutamic acid derivative can be present by forming ablock or can be present at random.

In the general formulas (I) and (II), R³ represents a hydrogen atom, aprotecting group, a hydrophobic group or a polymerizable group.Specifically, R³ may preferably be an acetyl group, an acryloyl group,or a methacryloyl group.

In the general formulas (I) and (II), R⁴ represents a hydroxyl group, anoxybenzyl group, a —NH—(CH₂)_(a)—X group, or an initiator residue.Herein, a represents an integer of 1-5, and X may preferably be theresidue of an amine compound comprising one or more of a primary,secondary, and tertiary amine, a quaternary ammonium salt, and aguanidino group, or may preferably be the residue of a compound otherthan amine. Furthermore, optionally R⁴ may preferably be —NH—R⁹ (R⁹represents an unsubstituted or a substituted straight or branched C₁₋₂₀alkyl group).

In the general formulas (I) and (II), m may be an integer of 5-2,000,preferably an integer of 5-270, and more preferably an integer of10-100. Also, n represents an integer of 2-5,000, y represents aninteger of 0-5,000, and preferably n and y may represent an integer of5-300, more preferably an integer of 10-100. Provided that y is notgreater than n.

While each of the repeating units in the general formulas (I) and (II)is represented according to the order of being specified for the sake ofconvenience of description, each of the repeating units may be presentat a random order. Specifically, it is preferred that each repeatingunit in the polyanionic segment can only be present at a random order asdescribed above.

While the molecular weight (Mw) of the block copolymers represented bythe general formulas (I) and (II) may not be specifically limited, itmay preferably be 3,000-30,000, more preferably 5,000-20,000. Forindividual segments, the molecular weight (Mw) of the PEG segment maypreferably be 500-15,000, more preferably 1,000-5,000, and the molecularweight (Mw) of the polyanionic segment may preferably be 500-50,000,more preferably 1,000-20,000.

While the method for producing the block copolymers represented by thegeneral formulas (I) and (II) may not be specifically limited, there canbe mentioned a method, for example, in which a segment (PEG segment)that contains the block part of R^(1a)o- or R^(1b)o- and a PEG chain issynthesized in advance, to one end (the end opposite to R^(1a)o- orR^(1b)o-) of the PEG segment, a predetermined monomer is polymerized inorder, and then the side chain is substituted or converted, as needed,so as to envelope the anionic group, or a method in which the above PEGsegment and a block part having a side chain containing an anionic groupare synthesized in advance, and then they are linked to each other. Theprocedure and condition of various reactions in the above productionmethods may be selected or designed as appropriate considering theconventional methods. The above PEG segment may be prepared using amethod for producing the PEG segment part of the block copolymerdescribed in, for example, WO96/32434, WO96/33233, WO97/06202, and thelike.

As a more specific method for producing the block copolymers representedby the general formula (I) and (II), there can be preferably mentioned amethod in which, for example, to the amino terminal of a PEG segmentderivative having an amino group at the end, a N-carboxylic acidanhydride (NCA) of a protective amino acid such as β-benzyl-L-aspartate(BLA) and Nε-Z-L-lysine is polymerized to synthesize a block copolymer,and then the side chain of each segment is substituted or converted tobecome a side chain having the above-mentioned anionic group.

As a specific example of block copolymers represented by the generalformulas (I) and (II), there may preferably be mentioned an anionicblock copolymer (hereinafter referred to as “PEG-P(Asp)” of thefollowing formula, comprising polyethylene glycol (hereinafter referredto as “PEG”), which is an uncharged hydrophilic segment, andpolyaspartic acid (hereinafter referred to as “P(Asp)”, which is ananionic segment (in the formulas hereinbelow, Na⁺ may be indicated as anexample of a counter ion, but the counter ion may not be limited to it).

wherein,

m represents an integer that indicates the degree of polymerization ofPEG,

n represents an integer that indicates the degree of polymerization ofP(Asp), and

any of a and b is greater than 0 and less than 1, provided that a+b=1.

As PEG-P(Asp), one that has a molecular weight (Mw) of the PEG segmentof 2,000 and a number (n in the above formula) of P(Asp) units of 70 or75 may be specifically preferred, P(Asp) being a polyanionic segment.

As each block copolymer of the above (A2) and (B1), for example, the onerepresented by the following general formula (III) and/or (IV) maypreferably be mentioned.

Wherein, in the structural formula of the general formulas (III) and(IV), the segments having a number (degree of polymerization) ofrepeating units of “m” are uncharged hydrophilic segments derived fromPEG (“PEG segments”), and the segments that combine the sections havinga number of repeating units of “n-y-z” and sections having a number ofrepeating units of “y” are cationic segments derived from polycations(hereinafter referred to as “polycationic segments”).

In the general formulas (III) and (IV), R^(1a) and R^(1b) represent,independently from each other, a hydrogen atom or an unsubstituted orsubstituted straight or branched C₁₋₁₂ alkyl group. As a straight orbranched C₁₋₁₂, there can be mentioned methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl,undecyl and the like. As a substituent when they are substituted, therecan be mentioned an acetalized formyl group, a cyano group, a formylgroup, a carboxyl group, an amino group, a C₁₋₆ alkoxycarbonyl group, aC₂₋₇ acylamido group, the same or different tri-C₁₋₆ alkylsiloxy group,a siloxy group, and a sillylamino group. As used herein acetalizationmeans the formation of an acetal section by the reaction of formylcarbonyl with 2 molecules of alkanol having 1-6 carbons or an optionallybranched alkylenediol having 2-6 carbons, and thus is also a method forprotecting the carbonyl group. For example, when the substituent is anacetalized formyl group, it can be hydrolyzed under an acidic mildcondition to be converted to another substituent, a formyl group (—CHO)(or an aldehyde group).

In the general formulas (III) and (IV), L¹ and L² represent a linkinggroup. Specifically, L¹ may preferably be —(CH₂)_(b)—NH— (b is aninteger of 1-5), and L² may preferably be —(CH₂)_(c)—CO— (c is aninteger of 1-5).

In the general formulas (III) and (IV), R^(2a), R^(2b), R^(2c) andR^(2d) represent, independently from each other, a methylene group or anethylene group. When both of R^(2a) and R^(2b) are a methylene group,they correspond to a poly(aspartic acid derivative), and when they arean ethylene group, they correspond to a poly(glutamic acid derivative),and when both of R^(2c) and R^(2d) are a methylene group, theycorrespond to a poly(aspartic acid derivative), and when they are anethylene group, they correspond to a poly(glutamic acid derivative). Inthese general formulas, when R^(2a) and R^(2b) (R^(2b) and R^(2a))represent both of a methylene group and an ethylene group, and whenR^(2c) and R^(2d) (R^(2d) and R^(2c)) represent both of a methylenegroup and an ethylene group, repeating units of the aspartic acidderivative and the glutamic acid derivative can be present by forming ablock or can be present at random.

In the general formulas (III) and (IV), R³ represents a hydrogen atom, aprotecting group, a hydrophobic group or a polymerizable group.Specifically, R³ may preferably be an acetyl group, an acryloyl group,or a methacryloyl group.

In the general formulas (III) and (IV), R⁴ represents a hydroxyl group,an oxybenzyl group, a —NH—(CH₂)_(a)—X group, or an initiator residue.Herein, a represents an integer of 1-5, and X may preferably be theresidue of an amine compound comprising one or more of a primary,secondary, and tertiary amine, a quaternary ammonium salt, and aguanidino group, or may preferably be the residue of a compound otherthan amine. Furthermore, optionally R⁴ may preferably be —NH—R⁹ (R⁹represents an unsubstituted or a substituted straight or branched C₁₋₂₀alkyl group).

In the general formulas (III) and (IV), R^(5a), R^(5b), R^(5c), andR^(5d) represent, independently from each other, a hydroxyl group, anoxybenzyl group, or a —NH—(CH₂)_(a)—X group. Herein, a represents aninteger of 1-5, and X may preferably be the residue of an amine compoundcomprising one or more of a primary, secondary, and tertiary amine, aquaternary ammonium salt, and a guanidino group, or may preferably bethe residue of a compound other than amine.

Among the total number of R^(5a) and R^(5b) and the total number ofR^(5c) and R^(5d), the presence of at least two —NH—(CH₂)_(a)—X groups(X represents (NH(CH₂)₂)_(e)—NH₂ (e is an integer of 0-5)) may bepreferred, the presence of 50% or more of the above total number may bemore preferred, and the presence of 85% or more of the above totalnumber may be even more preferred.

Also, all or part of R^(5a), R^(5b), R^(5c), and R^(5d) may preferablybe —NH—(CH₂)_(a)—X group (wherein a represents 2 and X represents(NH(CH₂)₂)_(e)—NH₂ (provided e is 1)).

Furthermore, in the —NH—(CH₂)_(a)—X group illustrated as R⁴, R^(5a),R^(5b), R^(5c), and R^(5d), X selected from the groups represented byeach of the following formulas may be most preferred.

In each of the above formulas, X² represents a hydrogen atom, a C₁₋₆alkyl group or an amino C₁₋₆ alkyl group, R^(7a), R^(7b), and R^(7c),independently from each other, represents a hydrogen atom or a methylgroup, d1, d2, and d3, independently from each other, represent aninteger of 1-5, e1, e2, and e3, independently from each other, representan integer of 1-5, f represents an integer of 0-15, g represents aninteger of 0-15, and R^(8a) and R^(8b), independently from each other,represent a hydrogen atom or a protecting group. Herein, the protectinggroup may preferably be selected from the group consisting of a Z group,a Boc group, an acetyl group, and a trifluoroacetyl group that areusually used as a protecting group for an amino group.

In the general formulas (III) and (IV), R^(6a) and R^(6b), independentlyfrom each other, represent a hydrogen atom, —C(═NH)NH₂, or a protectinggroup, wherein the protecting group may preferably be selected from thegroup consisting of a Z group, a Boc group, an acetyl group, and atrifluoroacetyl group that are usually be used a protecting group for anamino group. Also in the general formulas (III) and (IV), t maypreferably represent an integer of 2-6, more preferably 3 or 4.

In the general formulas (III) and (IV), m represents an integer of5-2,000, preferably an integer of 5-270, and more preferably an integerof 10-100. n represents an integer of 2-5,000, y represents an integerof 0-5,000, and z represents an integer of 0-5,000. n may preferablyrepresent an integer of 5-300, more preferably represent 0 or an integerof 10-100. y and z may preferably represent 0 or an integer of 5-300,more preferably 0 or an integer of 10-100. Provided that the sum of yand z (Y+z) is not greater than n.

While each of the repeating units in the general formulas (III) and (IV)is represented according to the order of being specified for the sake ofconvenience of description, each of the repeating units may be presentat a random order. Specifically, it is preferred that each repeatingunit in the polycationic segment can only be present at a random orderas described above.

While the molecular weight (Mw) of the block copolymers represented bythe general formulas (III) and (IV) may not be specifically limited, itmay preferably be 23,000-45,000, more preferably 28,000-34,000. Forindividual segments, the molecular weight (Mw) of the PEG segment maypreferably be 500-15,000, more preferably 1,000-5,000, and the molecularweight (Mw) of the polycationic segment may preferably be altogether500-50,000, more preferably 1,000-30,000.

While the method for producing the block copolymers represented by thegeneral formulas (III) and (IV) may not be specifically limited, therecan be mentioned a method, for example, in which a segment (PEG segment)that contains the block parτ of R^(1a)o- or R^(1b)o- and a PEG chain issynthesized in advance, to one end (the end opposite to R^(1a)o- orR^(1b)o-) of the PEG segment, a predetermined monomer is polymerized inorder, and then the side chain is substituted or converted, as needed,so as to envelope the cationic group, or a method in which the above PEGsegment and a block part having a side chain containing a cationic groupare synthesized in advance, and then they are linked to each other. Theprocedure and condition of various reactions in the above productionmethods may be selected or designed as appropriate considering theconventional methods. The above PEG segment may be prepared using amethod for producing the PEG segment part of the block copolymerdescribed in, for example, WO96/32434, WO96/33233, WO97/06202, and thelike.

As a more specific method for producing the block copolymers representedby the general formulas (III) and (IV), there can be preferablymentioned a method in which, for example, to the amino terminal of a PEGsegment derivative having an amino group at the end, a N-carboxylic acidanhydride (NCA) of a protective amino acid such as β-benzyl-L-aspartate(BLA) and Nε-Z-L-lysine is polymerized to synthesize a block copolymer,and then the side chain of each segment is substituted with diethylenetriamine (DET) etc. or converted to become a side chain having theabove-mentioned cationic group.

As a specific example of block copolymers represented by the generalformulas (III) and (IV), there may preferably be mentioned a cationicblock copolymer (hereinafter referred to as “PEG-P(Asp-AP)” of thefollowing formula comprising polyethylene glycol (hereinafter referredto as “PEG”), which is an uncharged hydrophilic segment, andpoly(diaminopentane structure-containing aspartic acid derivative)(hereinafter referred to as “P(Asp-AP)”, which is a cationic segment (inthe formula hereinbelow, Cl⁻ may be indicated as an example of a counterion, but the counter ion may not be limited to it).

wherein,

m represents an integer that indicates the degree of polymerization ofPEG,

n represents an integer that indicates the degree of polymerization ofP(Asp-AP), and

any of a and b is greater than 0 and less than 1, provided that a+b=1.

As PEG-P(Asp-AP), one that has a molecular weight (Mw) of the PEGsegment of 2,000 and a number (n in the above formula) of P(Asp-AP)units of 70 or 75 may be specifically preferred, P(Asp-AP) being apolycationic segment.

As the polymer of the above (B2) (iv), for example, the one representedby the following general formulas (V) and (VI) may preferably bementioned. On the explanation of the following general formulas (V) and(VI), the explanation on the above-mentioned general formulas (I) and(II) (excluding the explanation on the PEG segment) may be similarlyapplied as appropriate.

As used herein, as a specific example of polymers represented by thegeneral formulas (V) and (VI), there may preferably be mentioned ananionic homopolymer (hereinafter referred to as “Homo-P(Asp)” of thefollowing formula, comprising polyaspartic acid (P(Asp)), which is ananionic segment.

wherein,

n represents an integer that indicates the degree of polymerization ofP(Asp), and

any of a and b is greater than 0 and less than 1, provided that a+b=1.

As Homo-P(Asp), one that has a number (n in the above formula) of P(Asp)units of 70 or 82 may specifically be preferred, P(Asp) being apolyanionic segment.

As each block copolymer of the above (B1) (ii), for example, the onerepresented by the following general formula (VII) and/or (VIII) maypreferably be mentioned. On the explanation of the general formulas(VII) and (VIII), the explanation on the above-mentioned generalformulas (III) and (IV) may be similarly applied as appropriate.

As used herein, as a specific example of polymers represented by thegeneral formulas (VII) and (VIII), there may preferably be mentioned acationic homopolymer (hereinafter referred to as “Homo-P(Asp-AP)”) ofthe following formula, comprising poly(diaminopentanestructure-containing aspartic acid derivative) (P(Asp-AP)), which is acationic segment.

wherein,

n represents an integer that indicates the degree of polymerization ofP(Asp-AP), and

any of a and b is greater than 0 and less than 1, provided that a+b=1.

As Homo-P(Asp-AP), one that has a number (n in the above formula) ofP(Asp-AP) units of 70 or 82 may specifically be preferred, P(Asp-AP)being a polycationic segment.

(III-3: Additional Membrane Component)

During the formation of a vacant vesicle, an additional membranecomponent can be added unless it prevents vesicle formation or reducesstability, in addition to the first polymer and the second polymer. Theother membrane component may not be specifically limited, and specificexamples thereof may include a charged polymer, a charged nanoparticleand the like.

As the charged polymer, there can be mentioned any charged polymer thathas one or more charged segment (a cationic segment or an anionicsegment) mentioned above and that does not correspond to the firstpolymer or the second polymer.

As the charged nanoparticle, a metal-based nanoparticle having anelectric charge on the surface etc. may be mentioned.

As the other membrane component mentioned above, a single component maybe used alone, or two or more components may be used in any combinationor ratio.

Also while the amount used of the other membrane component mentionedabove may not be limited, the amount may preferably be kept roughly to alevel that does not prevent vesicle formation by the self-assembly ofthe first polymer and the second polymer. Specifically, relative to thetotal weight of the vesicle, the amount may usually be 30% or less,preferably 20% or less, and more preferably 10% or less.

(III-4: Method for Producing a Vacant Vesicle)

Since the vacant vesicle for use in the production method of the presentinvention may be formed using an electrostatic interaction between thefirst polymer and the second polymer, it can be easily produced bymixing the first polymer and the second polymer in an aqueous solution.Such a production method can produce the vesicle even without using anorganic solvent, and thus is advantageous in the field of DDS,biomaterials and the like.

Specifically, a first aqueous solution containing the first polymer anda second aqueous solution containing the second polymer are provided.The first and the second aqueous solutions may be purified by filtrationas desired.

The concentration of the first polymer in the first aqueous solution andthe concentration of the second polymer in the second aqueous solutionmay not be limited, and may be determined, as appropriate, byconsidering the ratio of the total charges of the first polymer and thesecond polymer, the solubility of the first polymer and the secondpolymer into the aqueous solutions, conditions such as the efficiency ofvesicle formation etc.

The type of the solvent for the first and the second aqueous solutionsmay not be limited, as long as it is an aqueous solvent. It maypreferably be water, but, as long as it does not prevent vesicleformation, there can be used a solvent having another component mixed inwater, such as physiological saline, an aqueous buffer, a mixed solventof water and a water soluble organic solvent etc. As an aqueous buffer,a 10 mM HEPES buffer etc. may be mentioned.

While the pH of the first and the second aqueous solutions may beadjusted as appropriate as long as it does not prevent vesicleformation, it may preferably be pH 5 or higher, more preferably pH 6.5or higher, and preferably pH 9 or lower, more preferably pH 7.5 orlower. pH can be easily adjusted by using a buffer solution as asolvent. The pH adjustment of the first and second aqueous solutions isadvantageous in maintaining the charged state of the first polymer andthe second polymer and efficiently forming a vesicle.

While the temperature of the first and second aqueous solutions can bedetermined as appropriate depending on the solubility in the solvent ofthe first polymer and the second polymer, it may preferably be 10° C. orhigher, more preferably 20° C. or higher, and preferably 80° C. orlower, more preferably 50° C. or lower.

While the ionic strength of the first and second aqueous solutions canbe adjusted as appropriate as long as it does not prevent vesicleformation, it may preferably be 0 mM or more, more preferably 10 mM ormore, and preferably 200 mM or less, more preferably 50 mM or less.

The above first and second aqueous solutions may be mixed to form avesicle. The mixing method is not limited, and the second aqueoussolution may be added to the first aqueous solution or the first aqueoussolution may be added to the second aqueous solution. Also, the firstand second aqueous solutions may be simultaneously placed in a vesselfor mixing. The mixture of the first and second aqueous solutions may beagitated as appropriate.

While the temperature at the time of mixing of the first and secondaqueous solutions is not limited as long as it does not prevent vesicleformation, it can be preferably determined by considering the solubilitydepending on the temperature of the first polymer and the secondpolymer. Specifically, it may preferably be 10° C. or higher, morepreferably 20° C. or higher, and preferably 60° C. or lower, morepreferably 50° C. or lower.

While, after mixing, the vacant vesicle formed may be immediatelysubjected to the production method of the present invention, time forleaving the mixture to stand may be allowed for in order to equalize thesystem. However, since the diameter of the vesicle formed may tend toincrease over time, it may usually be preferred to subject the vacantvesicle formed to the production method of the present invention withoutallowing for the standing time.

When another membrane component is used, said membrane component may bemixed with the first and second aqueous solutions. At this time, whilethis membrane component may be added to the first and second aqueoussolutions before mixing, there may preferably be no aggregation orinteraction that prevents vesicle formation between the membranecomponent and the first and second aqueous solutions. Also, the membranecomponent may be added simultaneously with the mixing of the first andsecond aqueous solutions, or the mixing of the first and second aqueoussolutions may be followed by the addition and further mixing of themembrane component. Another membrane component may be mixed as it is, oran aqueous solution containing the membrane component may be preparedand may be used in mixing. The conditions for preparing an aqueoussolution of the membrane component such as an aqueous solvent, pH,temperature, and ionic strength etc. are as described for the first andsecond aqueous solutions.

Furthermore, a procedure such as dialysis, dilution, concentration, andagitation may be added as appropriate.

IV: Encapsulation-Target Substance

The encapsulation-target substance for use in the production method ofthe present invention may not be specifically limited, and anysubstance-encapsulating vesicles can be selected as appropriatedepending on the uses and properties of the substance-encapsulatingvesicle.

Specifically, according to a simultaneous mixing method which is one ofthe conventional production method, there was a problem that when acharged substance was used as an encapsulation-target substance, vesicleformation by the self-assembly of polymers that are a membrane componentis prevented by the electric charge of the encapsulation-targetsubstance, and thus an appropriate substance-encapsulating vesicle couldnot be obtained. According to the production method of the presentinvention, however, there is no such limitation on the electricproperties of the encapsulation-target substance, and thus even when acharged substance was used as the encapsulation-target substance, asubstance-encapsulating vesicle can be efficiently formed.

Specifically, as the type of the encapsulation-target substance, therecan be mentioned a biomaterial, an organic compound, an inorganicsubstance and the like.

As the biomaterial, there can be mentioned protein, polypeptide, aminoacid, nucleic acid (DNA, RNA), lipid (fatty acid, glyceride, steroidetc.), carbohydrate (monosaccharide, polysaccharide), and a derivativethereof, as well as two or more of them bound together (glycoprotein,glycolipid, etc.). Among them, protein, carbohydrate etc. may bepreferred.

As the organic compound, there can be mentioned a light-emitting(fluorescent, phosphorescen) molecule, a water soluble drug, a watersoluble polymer, a water soluble molecular self-assembly (micelle,vesicle, nanogel, etc.) with a mean particle size of 100 nm or less, anemulsion with a mean particle size of 100 nm or less, and the like.Among them, a polymer micelle with a mean particle size of 50 nm or lessand a water soluble polymer with a molecular weight of 100,000 or lessmay be preferred.

As the inorganic substance, there can be mentioned a water-dispersiblemetal nanoparticle, an oxide nanoparticle (a silica nanoparticle, atitania nanoparticle, an iron oxide nanoparticle etc.), a semiconductornanoparticle (a quantum dot etc.), a water soluble carbon cluster, aboron cluster, a metal complex and the like. Among them, a quantum dotwith a mean particle size of 20 nm or less may be preferred.

As the encapsulation-target substance, by classifying by use, there canbe mentioned an anti-cancer agent (for example, a hydrophobicanti-cancer agent such as doxorubicin and paclitaxel, a metal complexanti-cancer agent such as cisplatin etc., and a polymer micellethereof), a gadolinium and iron compound used in diagnostic MRI etc., anorganic light-emitting (fluorescent, phosphorescen) dye, a quantum dotand the like.

While the molecular weight or particle size of the encapsulation-targetsubstance is not limited, the molecular weight of theencapsulation-target substance may usually be 200,000 or less,especially 100,000 or less, and the particle size of theencapsulation-target substance may usually be 100 nm or less, mostpreferably 50 nm or less, from the viewpoint of efficiently introducingan encapsulation-target substance into the vacant vesicle.

The ratio of the encapsulation-target substance used relative to thevacant vesicle may be adjusted according to the desired amount of theencapsulation-target substance as long as it does not destroy the vacantvesicle structure or prevent the encapsulation of theencapsulation-target substance into the vacant vesicle.

Only one type of the encapsulation-target substance may be used alone,or two or more types may be used in any ratio and combination.

V: Additional Step

While the production method of the present invention may only require astep of providing a vacant vesicle having a predetermined structure andmixing the vacant vesicle with an encapsulation-target substance in anaqueous medium, it may further have an additional step. Examples includetreatment with a cross-linker, filtration, dialysis, lyophilization etc.

Among them, when the substance-encapsulating vesicle is used in aphysiological environment or in the presence of a salt such asphysiological saline (for example, when used as a DDS), the formedsubstance-encapsulating vesicle may preferably be subjected to treatmentwith a cross-linker as a post-treatment, from the viewpoint ofpreventing an increase in particle size with time. Thus, in aphysiological environment or in the presence of a salt such asphysiological saline, the particle size of the vesicle having nocross-linker may tend to increase with time, but treatment with across-linker can prevent the increase in particle size.

While the type of the cross-linker is not limited, and may be selected,as appropriate, according to the use of the vesicle, the type of thefirst polymer and the second polymer, the type of another membranecomponent and the like, the cross-linker may preferably react withcharged groups (for example, a cationic group such as an amino group andan anionic group such as a carboxyl group) contained in the chargedsegment of the first polymer and the second polymer but not with theencapsulation-target substance, from the viewpoint of efficientlyconducting crosslinking and enhancing the stability of thesubstance-encapsulating vesicle. As a specific example of thecross-linker, there can be mentioned a cross-linker having an aminogroup (for example, glutaraldehyde, dimethyl suberimidatedihydrochloride (DMS), dimethyl 3,3′-dithiobispropionimidate (DTBP)), across-linker (for example, 1-ethyl-3-(3-dimethylaminopropy)carbodiimide(EDC)) that conducts crosslinking by fusing an amino group and acarboxyl group, with glutaraldehyde and EDC etc. being preferred and EDCbeing most preferred. While a single cross-linker may be used alone, twoor more types of crosslinking agent may be used in any combination orratio.

The amount used of a cross-linker is not limited, and may be selected,as appropriate, by considering the type of the cross-linker, the numberof crosslinking points, the amount of the component to be crosslinked,and the like. In the case of a cross-linker, for example, thatcrosslinks an amino group and a carboxyl group, the amount used maypreferably be selected so that the CL ratio defined by the followingequation (iii) can satisfy the conditions described below:

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 3} \right\rbrack & \; \\{\begin{matrix}{{CL}\mspace{14mu} {ratio}} \\\left( {{mole}\mspace{14mu} {ratio}} \right)\end{matrix} = \frac{\left\lbrack {{moles}\mspace{14mu} {of}\mspace{14mu} a\mspace{14mu} {cross}\text{-}{linker}} \right\rbrack}{\begin{bmatrix}{{moles}\mspace{14mu} {of}\mspace{14mu} {carboxyl}\mspace{14mu} {groups}} \\{{contained}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {first}\mspace{14mu} {polymer}} \\{{and}\mspace{14mu} {the}\mspace{14mu} {second}\mspace{14mu} {polymer}}\end{bmatrix}}} & {{Equation}\mspace{14mu} ({iii})}\end{matrix}$

From the viewpoint of efficiently conducting crosslinking and enhancingthe stability of the substance-encapsulating vesicle, the mass ratio ofthe cross-linker and the first and the second polymers may preferably beadjusted so as to give a CL ratio of usually 0.1 or more, preferably 0.5or more. On the other hand, when a substance-encapsulating vesicle isused as a DDS in drug delivery (for example, when used), the amount usedof the cross-linker may preferably be not too much, from the viewpointof allowing a drug to be efficiently released at the target site, andspecifically the mass ratio of the cross-linker and the first and thesecond polymers may preferably be adjusted so as to give a CL ratio ofusually 10 or less, preferably 5 or lower. However, the above range ofthe CL ratio is a rough measure, and in reality the CL ratio maypreferably be adjusted as appropriate according to the use of thevesicle, the type of the first polymer and the second polymer, the typeof the other membrane component and the like.

VI: Substance-Encapsulating Vesicle

In accordance with the production method of the present invention, asubstance-encapsulating vesicle that encapsulates anencapsulation-target substance in the cavity of the above vacant vesiclecan be obtained.

The substance-encapsulating vesicle produced by the production method ofthe present invention (hereinafter referred to as thesubstance-encapsulating vesicle of the present invention) comprises amembrane formed from a first polymer, which is a block copolymer havingan uncharged hydrophilic segment and a first charged segment, and asecond polymer, which has a second charged segment charged oppositely tothe first charged segment, a cavity surrounded by the above membrane,and a substance encapsulated in the cavity.

The structure of the membrane of the substance-encapsulating vesicle ofthe present invention is essentially equal to the structure of themembrane of the above-mentioned vacant vesicle. Thus, thesubstance-encapsulating vesicle of the present invention may preferablyhave a trilaminar structure similar to the structural membrane of thevacant vesicle explained using FIGS. 2, 3A-3B, and 4A-4B, and the shapethereof may usually be spherical or roughly spherical.

The particle size of the substance-encapsulating vesicle of the presentinvention may vary with the conditions such as the structure of thevacant vesicle, the type of the encapsulation-target substance, theenvironment surrounding the vesicle (the type of an aqueous medium),mixing etc., usually it may roughly be the same as the particle size ofthe vacant vesicle. Specifically the particle size of thesubstance-encapsulating vesicle of the present invention may preferablybe 10 nm or more, more preferably 50 nm or more, and preferably 1000 nmor less, more preferably 400 nm or less, and even more preferably 200 nmor less.

The membrane thickness of the substance-encapsulating vesicle of thepresent invention may also vary with the conditions such as thestructure of the vacant vesicle, the type of the encapsulation-targetsubstance, the environment surrounding the vesicle (the type of theaqueous medium), mixing etc., usually it may roughly be the same theparticle size and the membrane thickness of the vacant vesicle.Specifically the membrane thickness of the substance-encapsulatingvesicle of the present invention may preferably be 5 nm or more, morepreferably 10 nm or more, and preferably 30 nm or less, more preferably15 nm or less.

While, in the conventional methods, it was partly possible toencapsulate a substance in an electrostatically interacting vesicle(PICsome), though there was limitation in the range of application.Also, it was difficult or impossible to allow the substance to beencapsulated into the vesicle at a later time. In accordance with theproduction method of the present invention, it may become possible toproduce a substance-encapsulating and electrostatically interactingvesicle (PICsome) that was allowed to encapsulate a wide range ofsubstances in an electrostatically interacting vesicle (PICsome).

Since the substance-encapsulating vesicle of the present inventionstably retains various substances in the cavity of the vesicle formed bypolymer self-assembly, it can be effectively used as a DDS or in varioususes of functional materials having an active ingredient. For example,by using siRNA (small interfering RNA) as a constituent of anencapsulation-target substance or of the vesicle membrane, thesubstance-encapsulating vesicle obtained can be used as a DDS etc. forRNAi (RNA interference). It is also possible to allow a plurality ofdrugs to be encapsulated as an encapsulation-target substance, or to usethe vesicle in a combined drug therapy by using together as anencapsulation-target substance and a membrane material.

Since the substance-encapsulating vesicle of the present invention isnovel, and expands the range of application of PICsome with a meanparticle size of 100-200 nm exhibiting excellent blood retention andtumor accumulation, it is highly useful.

VII: Intra-Membrane Nucleic Acid-Containing Vesicle

In a vacant vesicle used in the production method of the presentinvention, the vesicle in which the first polymer is a block copolymerhaving an uncharged hydrophilic segment and a cationic segment (thus,the first charged segment is a cationic segment) and the second polymeris a polymer having nucleic acid as an anionic segment (thus, the secondcharged segment has nucleic acid as an anionic segment) is a vesiclehaving a novel structure in which the cavity is surrounded by a membranecomprising a predetermined block copolymer and nucleic acid. Such avesicle (hereinafter referred to as an “intra-membrane nucleicacid-containing vesicle”) per se is useful as a DDS for deliveringnucleic acid or as a biomaterial or functional material having nucleicacid as an active ingredient. Also, by using such an intra-membranenucleic acid-containing vesicle as a vacant vesicle in the aboveproduction method of the present invention, it can be made into asubstance-encapsulating vesicle that contains nucleic acid in themembrane and encapsulates another drug in the cavity, and thus may alsobe useful as a DDS for delivering nucleic acid and another drug incombination.

Now, such an intra-membrane nucleic acid-containing vesicle will beexplained below.

[VII-1: Structure of an Intra-Membrane Nucleic Acid-Containing Vesicle]

The structure of an intra-membrane nucleic acid-containing vesicle 10will be explained with reference to FIGS. 5A-5C. Any of FIGS. 5A-5C is aschematic diagram and the present invention is not limited to thesefigures in any way.

FIG. 5A is a partially broken view of a intra-membrane nucleicacid-containing vesicle 10. As shown in FIG. 5A, the intra-membranenucleic acid-containing vesicle 10 has a membrane 10 a and a cavity 10 bsurrounded by the membrane 10 a.

FIG. 5B is a partially broken enlarged view of the membrane 10 a of theintra-membrane nucleic acid-containing vesicle 10. As shown in FIG. 5B,the membrane 10 a has a trilaminar structure comprising an outer layer10 a _(o), an intermediate layer 10 a _(m), and an inner layer 10 a_(i), and mainly formed by a block copolymer 20 and nucleic acid 30.

FIG. 5C is an enlarged view of a block copolymer 20 and a nucleic acid30. As shown in FIG. 5C, the block copolymer 20 has an unchargedhydrophilic segment 20 a and a cationic segment 20 b.

It is believed, but not intended to be bound by a theory, that themechanism in which an intra-membrane nucleic acid-containing vesicle 10is formed from a block copolymer 20 and a nucleic acid 30 is as follows.Thus, when the block copolymer 20 and the nucleic acid 30 shown in FIG.5C are disposed in a system (for example, in an aqueous medium) that cangenerate the interaction of electric charges, they self-assemble, and,as shown in FIG. 5B, the positively charged segment 20 b and thenegatively charged nucleic acid 30 are electrostatically bound to forman intermediate layer 10 a _(m), and simultaneously an unchargedhydrophilic segment 20 a may be disposed outside thereof forming anouter layer 10 a _(o). Preferably, inside the intermediate layer 10 a_(m) as well, the uncharged hydrophilic segment 20 a is disposed to forman inner layer 10 a _(i). Thus, it is believed, the membrane 10 a of atrilaminar structure shown in FIG. 5A is thus formed, resulting in theformation of the intra-membrane nucleic acid-containing vesicle 10 shownin FIG. 5A.

Thus, according to a preferred embodiment, as shown in FIG. 5B, theuncharged hydrophilic segment 20 a forms the outer layer 10 a _(o) ofthe membrane 10 a, and the cationic segment 20 b and the nucleic acid 30electrostatically bind to form the intermediate layer 10 a _(m). Morepreferably, the uncharged hydrophilic segment 20 a mainly forms theinner layer 10 a _(i) of the membrane 10 a.

While the membrane 10 a of the intra-membrane nucleic acid-containingvesicle 10 may only comprise the block copolymer 20 and the nucleic acid30, it may contain another component in addition to the block copolymer20 and the nucleic acid 30, as long as the following structure may bemostly retained. The other component is not limited, and, for example, across-linker, a charged polymer, and a charged molecule etc. may bementioned. The cross-linker will be explained in detail below.

Also, since the intra-membrane nucleic acid-containing vesicle 10 mayusually be prepared in an aqueous medium as described below, and theinner layer 10 a _(i) of the membrane 10 a may preferably be composedmainly of the uncharged hydrophilic segment 20 a, as described below, anaqueous medium may usually be present in the cavity 10 b of theintra-membrane nucleic acid-containing vesicle 10 (thus, as used hereinthe cavity 10 b may sometimes be expressed as “internal water phase”).However, another substance may be present in the cavity 10 b.Specifically, when the intra-membrane nucleic acid-containing vesicle 10is used as a DDS etc., a drug may be contained in the cavity 10 b. Theembodiment of encapsulating a drug will be explained in detail below.

While the shape of the intra-membrane nucleic acid-containing vesicle 10may not be limited, it may be spherical or roughly spherical.

While the particle size of the intra-membrane nucleic acid-containingvesicle 10 may vary with the type and mass ratio of the block copolymer20 and the nucleic acid 30, the presence of a cross-linker, theenvironment (the type of an aqueous medium) surrounding theintra-membrane nucleic acid-containing vesicle 10 etc., it maypreferably be 10 nm or more, more preferably 50 nm or more, andpreferably 200 nm or less, more preferably 150 nm or less. In aphysiological environment or in the presence of a salt such asphysiological saline, as exemplified in the Example, the particle sizeof the intra-membrane nucleic acid-containing vesicle 10 having nocross-linker may tend to increase with time, but by introducing across-linker, an increase in particle size can be prevented.

While the membrane thickness of the intra-membrane nucleicacid-containing vesicle 10 a may vary with the type and mass ratio ofthe block copolymer 20 and the nucleic acid 30, the presence of across-linker, the environment (the type of the aqueous medium)surrounding the intra-membrane nucleic acid-containing vesicle 10 etc.,it may preferably be 5 nm or more, more preferably 10 nm or more, andpreferably 30 nm or less, more preferably 20 nm or less.

[VII-2: Block Copolymer]

A block copolymer used in an intra-membrane nucleic acid-containingvesicle has an uncharged hydrophilic segment and a cationic segment.

(VII-2a: Uncharged Hydrophilic Segment)

Details of an uncharged hydrophilic segment used in the intra-membranenucleic acid-containing vesicle are identical to the details on theuncharged hydrophilic segment used in the vacant vesicle explained inthe above section (III-2a: Uncharged hydrophilic segment).

(VII-2b: Cationic Segment)

Details of a cationic segment used in the intra-membrane nucleicacid-containing vesicle are identical to the details on the cationicsegment used in the vacant vesicle explained in the above section(III-2b-1: Cationic segment).

(VII-2c: Combination of an Uncharged Hydrophilic Segment and a CationicSegment)

The combination of an uncharged hydrophilic segment and a cationicsegment used in the intra-membrane nucleic acid-containing vesicle isnot limited, and any uncharged hydrophilic segment and any cationicsegment can be combined.

The numbers of the uncharged hydrophilic segment and the cationicsegment are also arbitrary, and may each be one or more, and in the caseof more than one, they may be the same or different. Usually, onecationic segment may preferably be bound relative to one unchargedhydrophilic segment. However, from the viewpoint of retaining a largeamount of nucleic acid in the vesicle, an embodiment in which more thanone cationic segment is bound to one uncharged hydrophilic segment mayalso be preferred.

The binding form of an uncharged hydrophilic segment and a cationicsegment is not limited, and they may be directly bound or may be boundvia a linking group.

As an example of a linking group, a hydrocarbon group having a valencycorresponding to the total number of the uncharged hydrophilic segmentsand the cationic segments may be mentioned. The hydrocarbon group as alinking group may be aliphatic or aromatic, or a linked body thereof.When it is aliphatic, it may be saturated or unsaturated, and may bestraight or branched or circular. While the molecular weight of ahydrocarbon group as a linking group is not limited, it may usually be5000 or less, preferably 1000 or less. As an example of a hydrocarbongroup as a linking group, a gallic acid derivative, a 3,5-dihydroxybenzoic acid derivative, a glycerin derivative, a cyclohexanederivative, a L-lysine etc. may be mentioned with a 3,5-dihydroxybenzoic acid derivative being preferred.

As another example of a linking group, a disulfide group may bementioned. The disulfide group is used to link one uncharged hydrophilicsegment and one cationic segment. By linking an uncharged hydrophilicsegment and a cationic segment via a disulfide group, it becomespossible to break up the disulfide group by the environment in which thevesicles were placed or by an external action to change the shape andproperty of the vesicle. By using this, for example when a vesicle isused as a DDS for delivering nucleic acid, it is thought to becomepossible to break up the disulfide group in vivo, thereby promoting therelease of nucleic acid constituting the vesicle membrane or a drug (theembodiment will be explained below) encapsulated in the vesicle.

While the ratio of the uncharged hydrophilic segment and the cationicsegment is also arbitrary, it is preferred that the molecular weightratio of the uncharged hydrophilic segment contained in the vesicle maybe brought into a predetermined range, from the viewpoint of promotingthe self-assembly of a block copolymer and nucleic acids and efficientlyproducing a uniform vesicle. A specific ratio will be explained below inthe section of (VII-3: Nucleic acid) since it may be preferred todetermine considering the amount of nucleic acid.

(VII-2d: Specific Example of a Block Copolymer)

As a preferred specific example of a block copolymer used in theintra-membrane nucleic acid-containing vesicle, one represented by thefollowing formula (IX) may be mentioned:

In the formula (IX),

R¹⁰ represents —(CH₂)₃NH₂, —(CH₂)₂NHC(═NH) NH₂, or —CONH(CH₂)_(s)—X,

[wherein,

s represents an integer of 0-20,

X is selected from the group consisting of —NH₂, a pyridyl group, amorpholyl group, a piperazinyl group, a 1-imidazolyl group, a 4-(C₁₋₆alkyl)-piperazinyl group, a 4-(amino C₁₋₆ alkyl)-piperazinyl group, apyrrolidine-1-yl group, a N-methyl-N-phenylamino group, a piperidinylgroup, a diisopropylamino group, a dimethylamino group, a diethylaminogroup, —(CH₂)_(t)NHR⁶⁰, and —(NR⁵⁰ (CH₂)_(o))_(p)NHR⁶⁰

(wherein,

R⁵⁰ represents a hydrogen atom or a methyl group,

R⁶⁰ represents a hydrogen atom, an acetyl group, a trifluoroacetylgroup, a benzyloxycarbonyl group, or a tert-butoxycarbonyl group, or aguanidino group,

o represents an integer of 1-5,

p represents an integer of 1-5, and

t represents an integer of 0-15)],

R²⁰ represents a hydrogen atom, an acetyl group, a trifluoroacetylgroup, an acryloyl group or a methacryloyl group, a cholesterolderivative group, an acyl group having 3-13 carbons, or a guanidinogroup (—C(═NH)NH₂),

R³⁰ represents a hydrogen atom, or an optionally substituted C₁₋₁₂ alkylgroup,

R⁴⁰ represents —(CH₂)_(g)R⁷⁰NH— (g represents an integer of 0-12)wherein R⁷⁰ represents a straight or branched C₁₋₁₂ alkyl,

a is an integer of 0-5,000

b is an integer of 0-5,000, provided that a+b is 2-5,000,

c is an integer of 0-20,

d is 0 or an integer satisfying c-1, and

e is an integer of 5-2,500.

In the formula (IX), when R¹⁰ represents —CONH(CH₂)_(s)—X, X may be thesame functional group for each repeating unit of the block copolymer ormay be a different functional group.

Among them, as the block copolymer of the present invention, one inwhich, in the formula (IX), R¹⁰ represents a —CONH(CH₂)_(s)—NH₂ group, srepresents an integer of 2-5, R²⁰ represents a hydrogen atom, R³⁰represents a methyl group, a represents an integer of 0-200, brepresents an integer of 0-200 provided that a+b represents 10-200, ande represents an integer of 10-300 may be specifically preferred.

(VII-3: Nucleic Acid)

A nucleic acid used in an intra-membrane nucleic acid-containing vesicleis not limited and may be selected, as appropriate, according to the useand property of the intra-membrane nucleic acid-containing vesicle.

Thus, the nucleic acid may be single-stranded or double-stranded, andmay be DNA or RNA. The presence or absence of protein coding or otherfunctions is not limited, either. Considering the use of the vesicle,however, it may preferably be a functional nucleic acid. As a functionalnucleic acid, siRNA, miRNA (micro RNA), antisense RNA, antisense DNA,ribozyme, DNA enzyme etc. can be mentioned. They are selected accordingto the use of the vesicle. For example, when the vesicle is used as aDDS for RNAi, siRNA is used as the nucleic acid.

The nucleic acid may be modified. As examples of a modified nucleicacid, a nucleic acid to which a hydrophobic functional group such ascholesterol or vitamin E has been bound may be mentioned for use invesicle stabilization etc.

The number of bases of the nucleic acid may not be specifically limited,and the range of usually 9 or more, preferably 12 or more, and morepreferably 19 or more, and usually 100 or less, preferably 40 or less,and more preferably 27 or less may be preferred. Specifically when siRNAis used as the nucleic acid, the number of bases is as described above,and usually 19-27, preferably 21-23.

One type of the nucleic acid may be used alone, or two or more may beused in any combination or ratio.

The amount used of the nucleic acid is not limited, and, from theviewpoint of promoting the self-assembly of a block copolymer and anucleic acid to efficiently produce a uniform vesicle, the amount usedmay preferably be selected so that the N⁺/P ratio defined in thefollowing equation (iv) may satisfy the condition explained below.

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 4} \right\rbrack & \; \\{\begin{matrix}{N^{+}\text{/}P\mspace{14mu} {ratio}} \\\left( {{mole}\mspace{14mu} {ratio}} \right)\end{matrix} = \frac{\begin{bmatrix}{{moles}\mspace{14mu} {of}\mspace{14mu} {protonated}\mspace{14mu} {amino}} \\{{groups}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {block}\mspace{14mu} {copolymer}}\end{bmatrix}}{\begin{bmatrix}{{moles}\mspace{14mu} {of}\mspace{14mu} {phosphate}\mspace{14mu} {in}} \\{{the}\mspace{14mu} {nucleic}\mspace{14mu} {acid}}\end{bmatrix}}} & {{Equation}\mspace{14mu} ({iv})}\end{matrix}$

wherein, while the moles of protonated amino groups in the blockcopolymer represents a value dependent on the structure of the cationicsegment of the block copolymer, it can be determined by a commonpotentiometric (acid/base) titration.

The value of N⁺/P ratio may be usually in the range of greater than 1.0,preferably 1.05 or more, more preferably 1.1 or more, and usually lessthan 3.0, preferably 2.8 or less, more preferably 2.5 or less. Thus themass ratio of the nucleic acid and the block copolymer may preferably beadjusted so that the value of the N⁺/P ratio may satisfy the aboverange.

However, the preferred value of the N⁺/P ratio may differ depending onvarious conditions. For example, when an unmodified nucleic acid is usedas the nucleic acid, the value of the N⁺/P ratio may be in the range ofusually greater than 1.0, preferably 1.05 or more, more preferably 1.1or more, and usually less than 1.5, preferably 1.4 or lower. On theother hand, when a modified nucleic acid (for example acholesterol-modified nucleic acid) is used as the nucleic acid, apreferred value of the N⁺/P ratio may be in a relatively high range.Thus the mass ratio of the nucleic acid and the block copolymer maypreferably be adjusted considering the above conditions.

The molecular weight of an uncharged hydrophilic segment may preferablybe decided considering the molecular weight of a cationic segment andnucleic acid that satisfy the above range of the N⁺/P ratio.Specifically, it is preferred that the molecular weight ratio X of theuncharged hydrophilic segment defined by the following equation (v) maybe set in the range of usually 0.01 or more, preferably 0.05 or more,and usually layer 0.35 or less, preferably 0.1 or less.

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 5} \right\rbrack & \; \\{X = \frac{M_{N}}{M_{N} + M_{C} + {M_{A}*\frac{P_{C}}{\left( {N^{+}/P} \right)*P_{A}}}}} & {{Equation}\mspace{14mu} (v)}\end{matrix}$

In the equation (ii),

M_(N) represents the molecular weight of the uncharged hydrophilicsegment,

M_(C) represents the molecular weight of the cationic segment,

M_(A) represents the molecular weight of the nucleic acid,

P_(C) represents the number of the cationized amino groups in thecationic segment, and

P_(A) represents the number of the phosphate groups in the nucleic acid.

(VII-4: Cross-Linker)

A cross-linker is not indispensable for an intra-membrane nucleicacid-containing vesicle, but when the intra-membrane nucleicacid-containing vesicle is used in a physiological environment or in thepresence of a salt such as physiological saline (for example, when usedas a DDS), a cross-linker may preferably be used as a membranecomponent, from the viewpoint of preventing an increase in particle sizewith time.

While the type of the cross-linker is not limited, and may be selectedas appropriate according to the use of the intra-membrane nucleicacid-containing vesicle and the type of the block copolymer and nucleicacid, the cross-linker may preferably react with the cationic group (forexample an amino group) contained in the cationic segment of the blockcopolymer and may not react with the nucleic acid, from the viewpoint ofefficiently conducting crosslinking and of enhancing the stability ofthe intra-membrane nucleic acid-containing vesicle obtained. As aspecific example of the cross-linker, there can be mentioned across-linker having an amino group (for example, glutaraldehyde,dimethyl suberimidate dihydrochloride (DMS), dimethyl3,3′-dithiobispropionimidate (DTBP),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), and a cross-linkerthat crosslinks phosphate groups (for example, a metal ion such as acalcium ion), with glutaraldehyde, DMS, DTBP etc. being preferred andglutaraldehyde being most preferred. While a single cross-linker may beused alone, two or more cross-linkers may be used in any combination orratio.

The amount used of a cross-linker is not limited, and may be selected,as appropriate, by considering the type of the cross-linker, the numberof crosslinking points, the amount of the component to be crosslinked,and the like. For example, in the case of a cross-linker that crosslinksamino groups, the amount used may preferably be selected so that theCL/N ratio defined by the following equation (vi) can satisfy thecondition described below:

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 6} \right\rbrack & \; \\{\begin{matrix}{{CL}\text{/}N\mspace{14mu} {ratio}} \\\left( {{mole}\mspace{14mu} {ratio}} \right)\end{matrix} = \frac{{Amount}\mspace{14mu} {expressed}\mspace{14mu} (a)}{\begin{matrix}{{Reference}\mspace{14mu} {amount}} \\{{expressed}\mspace{14mu} (b)}\end{matrix}}} & {{Equation}\mspace{14mu} ({vi})}\end{matrix}$

From the viewpoint of efficiently conducting crosslinking and enhancingthe stability of the intra-membrane nucleic acid-containing vesicleobtained, the mass ratio of the cross-linker and the block copolymer maypreferably be adjusted so as to give a CL/N ratio of usually 0.1 ormore, preferably 0.1 or more. On the other hand, when the intra-membranenucleic acid-containing vesicle is used in nucleic acid delivery (forexample, when used as a DDS for RNAi), the amount used of thecross-linker may preferably be not too much, from the viewpoint ofallowing the nucleic acid to be efficiently released at the target site,and specifically the mass ratio of the cross-linker and the blockcopolymer may preferably be adjusted so as to give a CL/N ratio ofusually 10 or less, preferably 7 or lower, and more preferably 5 orless.

(VII-5: Another Membrane Component)

While in producing an intra-membrane nucleic acid-containing vesicle,the addition of another membrane component different from the blockcopolymer or the nucleic acid to the membrane is not indispensable, theother membrane component can be added unless it prevents the formationof the intra-membrane nucleic acid-containing vesicle or reducesstability. The other membrane component may not be specifically limited,and specific examples thereof may include a charged polymer, a chargednanoparticle and the like.

As the charged polymer, a charged polyamino acid or a derivative thereofmay be preferred, and a cationic polyamino acid or a derivative thereofmay be most preferred. As the cationic polyamino acid or a derivativethereof, there can be mentioned polyaspartamide, polyglutamide,polylysine, polyarginine, polyhistidine, and a derivative thereof, witha polyaspartamide derivative and a polyglutamide derivative beingpreferred. While the number of the cationic polyamino acid or aderivative thereof is not limited, it may preferably be in the range of5 or more, more preferably 30 or more, and preferably 200 or less, morepreferably 100 or less.

As the charged nanoparticle, a metal-based nanoparticle etc. having anelectric charge on the surface can be mentioned.

One type of the above other membrane component may be used alone, or twoor more thereof may be used in any combination or ratio.

While the amount used of another membrane component is not limited, itmay preferably be kept to a degree that does not prevent vesicleformation by the self-assembly of the block copolymer and the nucleicacid. Specifically, relative to the total weight of the vesicle, it maybe usually 30% or less, preferably 20% or less, and more preferably 10%or less.

(VII-6: Method for Producing an Intra-Membrane Nucleic Acid-ContainingVesicle)

Since an intra-membrane nucleic acid-containing vesicle is formed usingan electrostatic interaction between a block copolymer and a nucleicacid, it can be easily produced by mixing a block copolymer and anucleic acid in an aqueous medium. According to such a productionmethod, the intra-membrane nucleic acid-containing vesicle can beproduced without using an organic solvent, and thus is advantageous inthe fields of DDS, biomaterials and the like.

Specifically, a first aqueous solution containing a block copolymer anda second aqueous solution containing a nucleic acid are provided. Thefirst and second aqueous solutions may be purified by filtration etc. asdesired.

The concentration of the block copolymer in the first aqueous solutionand the concentration of the nucleic acid in the second aqueous solutionmay not be limited, and may be determined, as appropriate, byconsidering the ratio of the total charged numbers of the blockcopolymer and the nucleic acid, the solubility of the block copolymerand the nucleic acid into the aqueous solutions, conditions such as theefficiency of forming a intra-membrane nucleic acid-containing vesicleetc.

The type of the solvent for the first and the second aqueous solutionsmay not be specifically limited as long as it is an aqueous solvent. Itmay preferably be water, but, a solvent having another component mixedin water, such as physiological saline, an aqueous buffer, and a mixedsolvent of water and a water soluble organic solvent etc. can also beused, as long as it does not prevent the formation of the intra-membranenucleic acid-containing vesicle. As an aqueous buffer, a 10 mM HEPESbuffer etc. may be mentioned.

While the pH of the first and the second aqueous solutions may beadjusted as appropriate as long as it does not prevent the formation ofa intra-membrane nucleic acid-containing vesicle, it may preferably bepH 5 or higher, more preferably pH 6.5 or higher, and preferably pH 9 orlower, more preferably pH 7.5 or lower. pH can be easily adjusted byusing a buffer solution as a solvent. The pH adjustment of the first andsecond aqueous solutions to be used is advantageous in maintaining thecharged state of the block copolymer and the nucleic acid andefficiently forming a vesicle.

While the temperature of the first and second aqueous solutions can bedetermined as appropriate depending on the solubility of the blockcopolymer and the nucleic acid in the solvent, it may preferably be 10°C. or higher, more preferably 20° C. or higher, and preferably 80° C. orlower, more preferably 60° C. or lower.

While the ionic strength of the first and second aqueous solutions canbe adjusted as appropriate as long as it does not prevent the formationof a intra-membrane nucleic acid-containing vesicle, it may preferablybe 5 mM or more, more preferably 10 mM or more, and preferably 300 mM orless, more preferably 150 mM or less.

The above first and second aqueous solutions may be mixed to form anintra-membrane nucleic acid-containing vesicle. The mixing method is notlimited, and the second aqueous solution may be added to the firstaqueous solution or the first aqueous solution may be added to thesecond aqueous solution. Also, the first and second aqueous solutionsmay be simultaneously placed in a vessel for mixing. The mixtureobtained of the first and second aqueous solutions may be agitated asappropriate.

While the temperature of the first and second aqueous solutions is notlimited as long as it does not prevent the formation of theintra-membrane nucleic acid-containing vesicle, it can be preferablydetermined by considering the solubility of the block copolymer and thenucleic acid corresponding to their temperature. Specifically, it maypreferably be 10° C. or higher, more preferably 20° C. or higher, andpreferably 60° C. or lower, more preferably 50° C. or lower.

While, after mixing, the intra-membrane nucleic acid-containing vesicleformed may be immediately subjected to the desired use, time for leavingthe mixture to stand may be allowed for in order to equalize the system.While the time for leaving the mixture to stand may vary with thecondition such as the efficiency of forming the intra-membrane nucleicacid-containing vesicle, it may preferably be 50 hours or less, morepreferably 30 hours or less. However, since the diameter of theintra-membrane nucleic acid-containing vesicle formed may tend toincrease with time, it may usually be preferred not to allow the leavingtime.

When a cross-linker is used, the cross-linker may be added to and mixedwith the mixture of the above first and second aqueous solutions. Thecross-linker may be mixed as it is, or an aqueous solution containingthe cross-linker may be prepared and this may be used in mixing. Theconditions for preparing an aqueous solution of the cross-linker such asan aqueous solvent, pH, temperature, and ionic strength are the same asdescribed for the first and second aqueous solutions.

When another membrane component other than the cross-linker is used, themembrane component may be added to and mixed with the above first andsecond aqueous solutions. At this time, while the membrane component maybe added to the first and second aqueous solutions before mixing, theremay preferably be no aggregation or interaction that prevents theformation of the intra-membrane nucleic acid-containing vesicle betweenthe membrane component and the first and second aqueous solutions. Also,the membrane component may be added simultaneously with the mixing ofthe first and second aqueous solutions, or the mixing of the first andsecond aqueous solutions may be followed by the addition and furthermixing of the membrane component. The other membrane component may bemixed as it is, or an aqueous solution containing the membrane componentmay be prepared and may be used in mixing. The conditions for preparingan aqueous solution of the membrane component such as an aqueoussolvent, pH, temperature, and ionic strength are the same as describedfor the first and second aqueous solutions.

Furthermore, a procedure such as dialysis, dilution, concentration, andagitation may be added as appropriate.

(VII-7: Use of an Intra-Membrane Nucleic Acid-Containing Vesicle)

Since the nucleic acid has been stably retained in the membrane in theintra-membrane nucleic acid-containing vesicle, the nucleic acid can beeffectively used as it is as a DDS for nucleic acid delivery or as afunctional material having the nucleic acid as an active ingredient. Forexample, by using siRNA as the nucleic acid, the intra-membrane nucleicacid-containing vesicle obtained can be used as a DDS for RNAi.

Also, a substance other than the nucleic acid, such as a drug, can beencapsulated in the cavity (internal water phase) of the intra-membranenucleic acid-containing vesicle. By so doing, the vesicle can beeffectively used as a DDS for delivering the combination of the nucleicacid and the drug. While the other drug is not limited, and may beselected as appropriate according to the use and property of thevesicle, there can be mentioned a protein, a peptide, an amino acid or aderivative thereof, a fat, a monosaccharide, an oligosaccharide, apolysaccharide, a glycoprotein, another drug etc. In classification byuse, there can be mentioned an cancer agent (for example, a hydrophobicanti-cancer agent such as doxorubicin and paclitaxel, a metal complexanti-cancer agent such as cisplatin), gadolinium and an iron compoundused in diagnostic MRI etc., an organic light-emitting (fluorescent,phosphorescen) dye, a quantum dot and the like.

When another drug is encapsulated in the cavity (internal water phase)of the intra-membrane nucleic acid-containing vesicle, the followingmethods may be mentioned:

(i) A method of adding the drug to the first and second aqueoussolutions and mixing before the formation of the intra-membrane nucleicacid-containing vesicle;

(ii) A method of adding the drug to the first and second aqueoussolutions and mixing during the formation the intra-membrane nucleicacid-containing vesicle; and

(iii) A method of, after mixing the first and second aqueous solutions,adding the drug to the aqueous solution containing the intra-membranenucleic acid-containing vesicle formed and mixing.

The methods of (i) and (ii) are useful when the drug is an uncharged(neutral) substance. However, the methods of (i) and (ii) are used for acharged (cationic or anionic) drug, the charged drug may inhibit theformation of the intra-membrane nucleic acid-containing vesicle, with aresult that no drug-encapsulating vesicle can be obtained.

On the other hand, the method of (iii) corresponds to the case of usingthe intra-membrane nucleic acid-containing vesicle as a vacant vesiclein the above method for producing a substance-encapsulating vesicle ofthe present invention. In accordance with the present method, asdescribed above, the drug can be used irrespective of whether it is anuncharged (neutral) substance or a charged (cationoic or anionic)substance. Though the reason for it is not clear, it is believed thatthe obtained intra-membrane nucleic acid-containing vesicle that oncedissociated has reassembled.

In the case of method (iii), the conditions are as described in theabove [II: Method for producing a substance-encapsulating vesicle], [IV:Encapsulation-target substance] and [V: Other steps].

While in any of the above methods of (i)-(iii), the drug to beencapsulated may be used as it is, an aqueous solution containing thedrug may be prepared and used in mixing. The conditions for preparingthe aqueous solution of the drug such as an aqueous solvent, pH,temperature, and ionic strength are the same as described above for thefirst and second aqueous solutions.

When another membrane component such as a cross-linker is used incombination, the order of mixing the drug and the membrane component isarbitrary. When a cross-linker is used, however, it may be preferred,from the viewpoint of efficiently encapsulating the drug into the cavityof the vesicle, that the encapsulation of the drug in the vesicle may befollowed by the addition and mixing of the cross-linker.

Furthermore, a procedure such as dialysis, dilution, concentration, andagitation may be added as appropriate.

(VII-8: Others)

While the intra-membrane nucleic acid-containing vesicle was explainedwith reference to specific embodiments as above, a person skilled in theart will be able to work the invention, based on the description of thepresent invention, by modifying the above embodiments as appropriate.

For example, in stead of the intra-membrane nucleic acid-containingvesicle explained in the embodiment, which is composed of a blockcopolymer having an uncharged hydrophilic segment and a cationic segmentand a nucleic acid, a copolymer having an uncharged hydrophilic segmentand a nucleic acid bound therein and a polymer comprising a cationicsegment may be substituted to form a similar intra-membrane nucleicacid-containing vesicle.

In this case as well, usually the cationic segment and the nucleic acidare electrostatically bound to form an intermediate layer, and anuncharged hydrophilic segment forms an outer layer and an inner layer,with a result that an intra-membrane nucleic acid-containing vesicle ofa structure comprising a membrane of a trilaminar structure and a cavitysurrounded thereby can be formed.

In such an embodiment, details of each component of the intra-membranenucleic acid-containing vesicle and details of the method for producingthe intra-membrane nucleic acid-containing vesicle are essentially thesame as the descriptions above. A person skilled in the art will be ableto work such an embodiment based on the description of the presentinvention by making necessary modifications as appropriate. Therefore,the method of producing an intra-membrane nucleic acid-containingvesicle having such a structure is also encompassed in the scope of thepresent invention.

EXAMPLES

Then, the present invention will be explained more specifically withreference to examples. The examples that follow are only forillustrative purposes and do not limit the present invention in any way.

In the following description, the term “solution” refers to a solution,unless otherwise specified, having a 10 mM phosphate buffer (pH 7.4) asa solvent.

Also in the following description, as the “vortex mixer”, Vortex-Genie 2manufactured by Scientific Industries Inc. was used unless otherwisespecified.

The mean particle size, the polydispersity index (PDI) and the zetapotential in the following description were measured with ZetasizerNano-ZS manufactured by Malvern unless otherwise specified.

Example Group I: Production and Evaluation of an Intra-Membrane NucleicAcid-Containing Vesicle

(I-a) Preparation of a Block Copolymer:

A block copolymer (hereinafter referred to as “PEG-PAsp(DAP)”)represented by the following formula having polyethylene glycol(molecular weight: about 2000) (hereinafter referred to as “PEG”) as anuncharged hydrophilic segment and a poly(diaminopentanestructure-containing aspartic acid derivative) (the degree ofpolymerization: 70) (hereinafter referred to as “PAsp(DAP)”) as acationic segment was synthesized.

wherein,

m represents the degree of polymerization of PEG, and is about 44,

n represents the degree of polymerization of PAsp(DAP), and is about 70,and

any of a and b is greater than 0 and less than 1, provided that a+b=1.

(I-b) Preparation of an Intra-Membrane Nucleic Acid-Containing Vesicle:

As siRNA, GL3 (the sense strand: 5′-CUU ACG CUG AGU ACU UCG AdTdT-3′(SEQ ID NO: 1), the antisense strand: 5′-UCG AAG UAC UCA GCG UAAGdTdT-3′ (SEQ ID NO: 2): the number of bases: 21) was used. To 50 μl ofa 1 mg/ml siRNA solution, a 1 mg/ml PEG-PAsp(DAP) aqueous solutionobtained in the above (I-a) was added at a volume that enables to obtaina value of the N⁺/P ratio defined in following Table 1, and agitated andmixed with a vortex mixer for 2 minutes to prepare the mixtures ofExamples I-1 to I-4 and Comparative Examples I-1 to I-4.

TABLE 1 N⁺/P ratio Example I-1 1.1 Example I-2 1.15 Example I-3 1.2Example I-4 1.4 Comparative Example I-1 0 Comparative Example I-2 1.0Comparative Example I-3 1.5 Comparative Example I-4 2.0

(I-c) Evaluation of an Intra-Membrane Nucleic Acid-Containing Vesicle:(I-c1) Measurement by Dynamic Light Scattering:

In order to investigate the physical properties of particles present inthe mixtures of Examples I-1 to I-4 and Comparative Examples I-1 to I-4,measurement by the dynamic light scattering method was conducted todetermine the mean particle size and the polydispersity index (PDI).

The measurement results of the mean particle size and the polydispersityindex are shown in FIGS. 6A and 6B, respectively. It can be seen fromFIG. 6A that in the range of N⁺/P ratio of greater than 1.0 and lessthan 1.5 (Examples I-1 to I-4), the mean particle size remains constantat about 100 nm, whereas at a N⁺/P ratio of 1.0 or less (ComparativeExamples I-1, I-2), the mean particle size becomes remarkably small, andat a N⁺/P ratio of 1.5 or more (Comparative Examples I-3, I-4),conversely, the mean particle size becomes remarkably large. Also, itcan be seen from FIG. 6B that in the range of N⁺/P ratio of greater than1.0 and less than 1.5 (Examples I-1 to I-4), the polydispersity indexbecomes small and uniform spherical particles are present. By takingthese results together, it can be seen that in the range of N⁺/P ratioof greater than 1.0 and less than 1.5 (Examples I-1 to I-4),PEG-PAsp(DAP) and siRNA self-assemble and therefore uniform sphericalparticles with a mean particle size of about 100 nm are formed in themixture.

(I-c2) Examination with a Transmission Electron Microscope:

The particles in the mixtures of Examples I-1 to I-4 and ComparativeExamples I-1 to I-4 that were obtained in the above (I-b) were examinedby a transmission electron microscope according to the followingprocedure.

Onto a Cu grid (Nisshin EM Corporation) that was carbon-coated afterattaching a collodion supporting membrane thereto, the mixture of theabove (I-b) was added dropwise, and furthermore a trace amount ofuranium acetate was added dropwise. Then excess water was blotted offwith a filter paper, and dried to prepare a specimen grid in which theparticles in the mixture of the above (I-b) were fixed. The specimengrid obtained was examined with a transmission electron microscope.

Examples of electron photomicrographs obtained for Example I-3 (N⁺/Pratio=1.2) and Comparative Example I-4 (N⁺/P ratio=2.0) are shown inFIGS. 7A and 7 b, respectively. Since a multitude of concentricparticles with a mean particle size of about 100 nm were observed in theelectron photomicrographs of Example I-2 (see FIG. 7A), it can be seenthat uniform spherical vesicles with a mean particle size of about 100nm having cavities were formed. On the other hand, in the electronphotomicrograph (see FIG. 7B) of Comparative Example I-4, no suchconcentric uniform particles were observed, and instead large particlesof irregular shape were only present.

For electron photomicrographs of the other N⁺/P ratios, in the range ofa N⁺/P ratio of greater than 1.0 and less than 1.5, a multitude ofconcentiric particles with a mean particle size of about 100 nm wereobserved as in FIG. 7A, whereas at a N⁺/P ratio of 1.0 or less or 1.5 ormore, such particles were not observed.

When these results are taken together with the measurement results ofthe above dynamic light scattering method, it can be seen that in therange of a N⁺/P ratio of greater than 1.0 and less than 1.5 (ExamplesI-1 to I-4), novel vesicles (intra-membrane nucleic acid-containingvesicles) having a membrane formed by PEG-PAsp(DAP) and siRNA wasformed.

Example Group II: Production and Evaluation of a CrosslinkedIntra-Membrane Nucleic Acid-Containing Vesicle (II-a) Preparation of aCrosslinked Intra-Membrane Nucleic Acid-Containing Vesicle

Vesicles were formed in a procedure similar to that described in “(I-b)Preparation of an intra-membrane nucleic acid-containing vesicle” ofExample group I except that the N+/P ratio was fixed at 1.4. Two hoursafter preparation, to 30 μl of the vesicle solution obtained, 10 μl of aglutaraldehyde solution with a concentration defined in the followingTable 2 was added as a cross-linker, and then agitated and mixed with avortex mixer for 2 minutes to prepare the vesicle-containing mixtures ofReference Example II and Examples II-1 to II-6. Each glutaraldehydesolution was prepared by diluting a 70% by weight glutaraldehyde(manufactured by Wako Pure Chemical Industries, Ltd.) with a buffersolution (HEPES buffer). The CL/N ratios of the vesicle-containingmixtures obtained finally are shown together in the following Table 2.

TABLE 2 Glutaraldehyde solution CL/N (% by weight) ratio ReferenceExample 0 0 II Example II-1 0.00875 1.0 Example II-2 0.0175 2.0 ExampleII-3 0.0875 10 Example II-4 0.175 20 Example II-5 0.875 100 Example II-61.75 200

(II-b) Evaluation of a Crosslinked Intra-Membrane NucleicAcid-Containing Vesicle:

The vesicle-containing mixture of Example II-1 obtained in the above(II-a) was examined by a phase-contrast cryo-transmission electronmicroscope. Specifically, a sample solution was developed onto amicrogrid, excess water was blotted off with a filter paper, and thenwas quick-frozen in a liquified ethane using EM CPC cryo-station (LeicaMicrosystems, Vienna, Austria) to prepare a specimen for examination.Images were photographed using a transmission electron microscopeJEM2011 (manufactured by JEOL Ltd.) equipped with a Zernike phase plate.The phase-contrast cryo-transmission electron micrographs are shown inFIGS. 8A-8D. All the vacant vesicles (crosslinked intra-membrane nucleicacid-containing vesicles) obtained had a similar structure and werespherical vesicles each comprising one dividing wall with a thickness ofabout 10 nm, and particle size was about 100 nm.

Also, to mixtures containing the intra-membrane nucleic acid-containingvesicles of Reference Example II and Examples II-1 to II-6 obtained inthe above (II-a), sodium chloride or a sodium chloride-containing buffersolution was added to a concentration equal to physiological saline (a150 mM sodium chloride solution) and mixed, and then after apredetermined time elapsed, they were measured by the dynamic lightscattering method to investigate changes with time in the mean particlesize and the polydispersity index (PDI).

Relationship between the mean particle size and the CL/N ratio and theelapsed time is shown in the graph of FIG. 9A, and the relationshipbetween the polydispersity index (PDI) and the CL/N ratio and theelapsed time is shown in the graph of FIG. 9B, respectively. From FIGS.9A and 9B, it can be seen that in the vacant vesicles (theintra-membrane nucleic acid-containing vesicles that are notcrosslinked) of Reference Example II (the CL/N ratio=0, i.e. nocross-linker) the particle size increased with time under aphysiological condition, whereas all the vacant vesicles (thecrosslinked intra-membrane nucleic acid-containing vesicles) of ExamplesII-1 to II-6 (the CL/N ratio ≧1, i.e. with a cross-linker) the particlesremained uniform and spherical particles with almost no changes withtime in particle size even under a physiological condition.

Example Group III: Evaluation of Efficiency of siRNA Introduction intothe Intra-Membrane Nucleic Acid-Containing Vesicle (III-a) Evaluation ofRNAi Activity: (III-a1) Preparation of an Intra-Membrane NucleicAcid-Containing Vesicle

The vesicle-containing mixtures of Examples III-1 to III-3 andReferences Example III-1 and III-2 were prepared in a procedure similarto that described in “(II-a) Preparation of the intra-membrane nucleicacid-containing vesicle” of Example group II, except that glutaraldehydesolutions with concentrations defined in the following Table 3 were usedas the cross-linker and the CL/N ratios of the vesicle-containingmixtures obtained finally were adjusted to give values defined in thefollowing Table 3.

TABLE 3 Glutaraldehyde solution CL/N (% by weight) ratio Example III-1 00 Example III-2 0.00875 1.0 Example III-3 0.0175 2.0 Reference Example0.0875 10 III-1 Reference Example 0.175 20 III-2

Also, the control mixtures corresponding to Examples III-1 to III-3 andReference Examples III-1 and III-2 were prepared in a procedure similarto the above procedure, except that a scramble (the sense strand: 5′-UUCUCC GAA CGU GUC ACG UdTdT-3′ (SEQ ID NO: 3), the antisense strand:5′-ACG UGA CAC GUU CGG AGA AdTdT-3′ (SEQ ID NO: 4): the number of bases:21) was used as the control siRNA.

(III-a2) Measurement of RNAi Activity:

Using mixtures containing the intra-membrane nucleic acid-containingvesicles of Examples III-1 to III-3 and Reference Examples III-1 andIII-2 obtained in the above (III-a1), the mouse melanoma cell B16F10-Lucwas treated in the following procedure to investigate the efficiency ofsiRNA introduction.

Thus, to the mouse melanoma cell B16F10-Luc in a DMEM medium containing400 μl of 10% bovine fetal serum, each of the mixtures (containing GL3as siRNA) of Examples III-1 to III-3 and Reference Examples III-1 andIII-2 was added to a siRNA-converted concentration of 500 nM, culturedfor 48 hours in an incubator, the culture medium was removed, and thecell lysate was added. To 20 μl of the supernatant, 100 μl of aluciferase assay solution (manufactured by Promega) was added, and theamount of light emitted was measured by a luminometer to determine theamount expressed of the luciferase gene.

Also, using the control mixtures (containing the scramble as siRNA)corresponding to Examples III-1 to III-3 and Reference Examples III-1and III-2, a similar procedure was conducted to determine the amountexpressed of the luciferase gene.

Furthermore, the cell group to which no siRNA was added was subjected toa similar procedure to determine the amount expressed of the luciferasegene.

For each of Examples III-1 to III-3 and Reference Examples III-1 andIII-2, using the amount expressed a and the reference amount expressed bdetermined in the above procedure, the relative amount expressed c wasdetermined from the following equation (vii):

$\begin{matrix}\left\lbrack {{Mathematical}\mspace{14mu} 7} \right\rbrack & \; \\{{{Relative}\mspace{14mu} {amount}\mspace{14mu} {expressed}\mspace{14mu} (c)\mspace{14mu} (\%)} = \frac{{Amount}\mspace{14mu} {expressed}\mspace{14mu} (a)}{\begin{matrix}{{Reference}\mspace{14mu} {amount}} \\{{expressed}\mspace{14mu} (b)}\end{matrix}}} & {{Equation}\mspace{14mu} ({viii})}\end{matrix}$

The measurement results of the relative amount expressed of theluciferase gene in each of Examples III-1 to III-3 and ReferenceExamples III-1 and III-2 are shown in FIG. 10. As shown in FIG. 10, theexpression of the luciferase gene was significantly suppressed by theintra-membrane nucleic acid-containing vesicles of Examples III-1 toIII-3 (the CL/N ratio is 0-2), RNAi activity was noted. Therefore, fromthe intra-membrane nucleic acid-containing vesicles of Examples III-1 toIII-3 (the CL/N ratio is 0-2), it can be seen that siRNA was efficientlyintroduced into the cell.

(III-b) Evaluation of Cell Uptake by Confocal Microscope (III-b1)Preparation of an Intra-Membrane Nucleic Acid-Containing Vesicle

The vesicle-containing mixtures of Examples III′-1 to III′-3 andReference Examples III′-1 to III′-4 were prepared in a manner similar tothat of the above (III-a1), except that Cy5-labelled G13 in which Cy5was attached to the 5′-end of the antisense strand and the sense strandof the above GL3 was used as siRNA, and a glutaraldehyde solution of theconcentration defined in the following Table 4 was used as across-linker, and the CL/N ratio of the vesicle-containing mixtureobtained finally was adjusted to give values defined in the followingTable 4.

TABLE 4 Glutaraldehyde solution CL/N (% by weight) ratio Example III′-10 0 Example III′-2 0.00875 1.0 Example III′-3 0.0175 2.0 ReferenceExample 0.0875 10 III′-1 Reference Example 0.175 20 III′-2 ReferenceExample 0.875 100 III′-3 Reference Example 1.75 200 III′-4(III-b2) Examination with a Confocal Microscope

Cell uptake by siRNA after cell treatment with the intra-membranenucleic acid-containing vesicle obtained was examined with a confocalmicroscope.

Thus, to mouse melanoma cell B16F19-Luc in a DMEM medium containing 1 mlof 10% fetal bovine serum, each of the mixtures (siRNA-convertedconcentration of 500 nM) of Examples III′-1 to III′-3 and ReferenceExamples III′-1 to III′-4 was added, and 24 hours later, examined with aconfocal microscope (LSM 510 META NLO manufactured by Carl Zeiss).

The confocal micrograph of Example III′-2 (the CL/N ratio=1.0) is shownin FIG. 11A. In FIG. 11A, the white regions indicate siRNA. From FIG.11A, it can be seen that siRNA has been markedly taken up into the cell.

Similarly, in Example III′-1 (the CL/N ratio=0) and Example III′-3 (theCL/N ratio=2.0) as well, the remarkable uptake of siRNA into the cellwas observed.

The confocal micrographs obtained for Examples III′-1 to III′-3 andReference Examples III′-1 to III′-4 were subjected to image analysis.Specifically, by counting the number of white pixels derived fromCy5-labelled siRNA, the amount of siRNA taken up into the cell wasestimated.

The result obtained is shown in the graph of FIG. 11B, It can be seenfrom FIG. 11B that the amount of siRNA taken up into the cell wassignificantly large in the intra-membrane nucleic acid-containingvesicle having a CL/N ratio of 0-2 and specifically in theintra-membrane nucleic acid-containing vesicle having a CL/N ratio of1.0.

The above results indicate that by the intra-membrane nucleicacid-containing vesicles of Examples III′-1 to III′-3 (the CL/N ratio is0-2), siRNA was efficiently taken up into the cell.

Example Group IV: Production and Evaluation of the UnchargedSubstance-Encapsulating Intra-Membrane Nucleic Acid-Containing Vesicle(IV-a) Production of n Uncharged Substance-Encapsulating Intra-MembraneNucleic Acid-Containing Vesicle

In the procedure described in “(III-b1) Preparation of a vesicle” of theExample group III, in addition to 47.8 μl of a 1 mg/ml aqueous solutionof PEG-PAsp(DAP) and 50 μl of a 1 mg/ml aqueous solution of siRNA (orCy3-labelled siRNA) (N+/P ratio: 1.4), 10 μl of a 10 mg/ml aqueoussolution of fluorescent dextran (manufactured by Aldrich, molecularweight 10000), which is an uncharged substance, was used as anencapsulation-target substance, and agitated and mixed with a vortexmixer in the order of “agitation/mixing A”→“agitation/mixing B” in thefollowing table to prepare the vesicles. Two hours after thepreparation, a 0.1875% by weight aqueous solution of glutaraldehyde wasadded as a cross-linker and further mixed (the CL/N ratio: 100) andpurified by an ultrafiltration membrane with a molecular weight cutoffof about 300,000 to prepare the vesicle-containing mixtures of ExamplesIV-1 to IV-3. Also, a similar procedure was conducted without usingfluorescent dextran to prepare a vesicle-containing mixture of ReferenceExample VI. Each of agitation/mixing A and B was conducted with a vortexmixer at about 3300 rpm for 2 minutes. Between agitation/mixing A andagitation/mixing B, and after agitation/mixing B, the leaving time of 3minutes or more was allowed, respectively.

TABLE 5 Mixing procedure Agitation/mixing A → Agitation/mixing B ExampleIV-1 PEG-PAsp(DAP) and → siRNA was added and fluorescent dextran furthermixed were agitated/mixed Example IV-2 siRNA and → PEG-PAsp(DAP) wasfluorescent dextran added and further were agitated/mixed mixed ExampleIV-3 PEG-PAsp(DAP) and → Fluorescent dextran siRNA were was added andagitated/mixed further mixed (vacant vesicle formation) ReferencePEG-PAsp(DAP) and fluorescent siRNA were Example IV mixed (vacantvesicle formation, without using fluorescent dextran)

(IV-b) Evaluation of an Uncharged Substance-Encapsulating Intra-MembraneNucleic Acid-Containing Vesicle

The vesicle-containing mixtures of Example IV-1 to IV-3 obtained in theabove (IV-a) were subjected to measurement by fluorescence correlationspectroscopy (FCS) to calculate the speed of motion of fluorescentparticles in a micro volume, i.e. the translational diffusioncoefficient. From changes in the translational diffusion coefficientobtained of the fluorescent dextran, it becomes possible to determinewhether the fluorescent dextran has been encapsulated into the vesicleor not.

Specifically, using an ultrafiltration membrane with a molecular weightcutoff of 300,000, each vesicle solution was ultrafiltrated for 5 timesat a condition of 2000 G, 8 minutes to remove the released fluorescentdextran. After the ultrafiltration procedure, each vesicle solution wasadjusted to a concentration of 100 μg/ml siRNA, and subjected to FCSmeasurement using a confocal fluorescent microscope (LSM510)manufactured by Carl Zeiss. The fluorescent dextran was excited by anargon laser (488 nm), the object lens used was a water immersion lenswith a 40-fold magnification, and analysis was performed by the Confocor3 software.

Also, as a control example, a 0.5 μg/ml solution of fluorescent dextranwas subjected to similar measurement to calculate a diffusion constant.

The result of the diffusion constant obtained is shown in FIG. 12. Asshown in FIG. 12, the diffusion constant of the vesicles of ExamplesIV-1 to IV-3 obtained by mixing PEG-PAsp(DAP), siRNA, and fluorescentdextran was much smaller than the diffusion constant of the controlexample of the fluorescent dextran, and was about the same level as thediffusion constant of the vesicle of Reference Example IV formed fromPEG-PAsp(DAP) and fluorescent siRNA.

The above results revealed that in Examples IV-1 to IV-3, irrespectiveof the order of mixing PEG-PAsp(DAP), siRNA, and fluorescent dextran,the intra-membrane nucleic acid-containing vesicle (a vacant vesicle)comprising PEG-PAsp(DAP) and siRNA was formed as in Reference ExampleVI, and the substance-encapsulating intra-membrane nucleicacid-containing vesicle containing fluorescent dextran (anencapsulation-target substance) in the cavity (internal water phase) wasobtained.

It can be seen, specifically, that by the procedure of forming anintra-membrane nucleic acid-containing vesicle (a vacant vesicle)comprising PEG-PAsp(DAP) and siRNA beforehand, then adding fluorescentdextran (an encapsulation-target substance) followed by agitating andmixing in Example IV-3, a substance-encapsulating intra-membrane nucleicacid-containing vesicle encapsulating the fluorescent dextran (anencapsulation-target substance) in the cavity (internal water phase)thereof was obtained. This is a surprising finding.

In Example IV-1 (after PEG-PAsp(DAP) and fluorescent dextran wereagitated and mixed, siRNA was added and further mixed) and Example IV-2(after siRNA and fluorescent dextran were agitated and mixed,PEG-PAsp(DAP) was added and further mixed) as well, similarsubstance-encapsulating intra-membrane nucleic acid-containing vesicleshave been obtained. That this was due to the use of fluorescent dextranwhich is an uncharged substance as an encapsulation-target substance isclear from the comparison with the following Example group V.

Example Group V: Production and Evaluation of a Negatively ChargedSubstance-Encapsulating Vesicle Example V-1: Production of a QD (aNegatively Charged Substance)-Encapsulating Vesicle

As the first polymer, an anionic block copolymer PEG-P(Asp) (zetapotential: −30.6 mV) comprising polyethylene glycol (PEG) (molecularweight: about 2000), which is an uncharged hydrophilic segment, andpolyaspartic acid (P(Asp)) (the degree of polymerization: 75), which isan anionic segment, was used.

As the second polymer, a cationic homopolymer Homo-P(Asp-AP) (zetapotential: +16.3 mV) comprising poly(diaminopentane structure-containingaspartic acid derivative) (P(Asp-AP)) (the degree of polymerization:82), which is a cationic segment, was used.

As the encapsulation-target substance, CdTe quantum dot (hereinafterreferred to as “QD”) (mean particle size: 4.2 nm; zeta potential: −64.1mV; reference: A. Zintchenko, et al. Molecular Therapy (2009) 17, 11,1849-1856) was used.

Each of the first polymer and the second polymer was dissolved in a 10mM phosphate buffer (pH 7.4) (aqueous medium) to a polymer concentrationof 2.0 mg/ml. The first polymer solution and the second polymer solutionobtained were placed in an Eppendorf tube so that the charge ratiobecomes equal (i.e. the C/A ratio=1.0) and mixed, and then agitated witha vortex mixer at about 3300 rpm for 2 minutes to obtain a solutioncontaining the vesicles (vacant vesicles) formed by the self-assembly ofthe first polymer and the second polymer.

The vacant vesicle-containing solution obtained as above was measured bythe dynamic light scattering method to determine pore size distribution,mean particle size, polydispersity index (PDI), and zeta potential. At amean particle size of 104 nm, the formation of monodispersion particleswas noted. The PDI was 0.045 and the zeta potential was −9.8 mV.

To the vacant vesicle-containing solution obtained as above, a solutionof the encapsulation-target substance was added to prepare a solution (asubject solution to be mixed) with a total polymer concentration of 1.0mg/ml and an encapsulation-target substance concentration of2.3×10¹⁵/ml. This subject solution to be mixed was agitated and mixedwith a vortex mixer at about 3300 rpm for 2 minutes, and then allowed tostand for over 3 minutes. The solution obtained after mixing wastransparent.

The solution after mixing was added to a solution (in a 10 mM phosphatebuffer (0 mM NaCl), pH 7.4) containing 10 equivalents of EDC (purchasedfrom PEPTIDE INSTITUTE, INC.) relative to the carboxyl group containedin PEG-P(Asp), and then allowed to stand at room temperature for 12hours to crosslink the polymer. QD remaining in the free state in thesolution without being encapsulated was removed by centrifugalultrafiltration (VIVASPIN 20 manufactured by Sartorius Sterium Biotech,a molecular weight cutoff of 300,000 was used; 300 rpm, 4° C.) to purifythe solution, which was then measured by the dynamic light scatteringmethod to determine particle size distribution, mean particle size, andpolydispersity index (PDI). The graph of particle size distribution isshown in FIG. 13. At a mean particle size of 114 nm, the formation ofmonodispersion particles was noted. The PDI was 0.065.

Subsequently, a fluorescence correlation spectroscopy (FCS) measurementwas conducted at room temperature using a confocal fluorescentmicroscope (LSM510) manufactured by Carl Zeiss. The fluorescent dextranwas excited by an argon laser (488 nm), the object lens used was a waterimmersion lens with a 40-fold magnification, and analysis was performedby the Confocor 3 software to determine an autocorrelation functionG(τ). The QD solution (2.3×10¹⁵/ml), which is an encapsulation-targetsubstance, was also subjected to MCS measurement to determine anautocorrelation function G(τ). The temporal attenuation curve of theautocorrelation function G(τ) obtained is shown in FIG. 14.

The autocorrelation function G(τ) is a function for evaluating thefluctuation of fluorescent intensity after time τ, and attenuates withtime and converges after a sufficient time. The longer the time (slowattenuation) until convergence, the more gradual the fluctuation is,i.e. indicates that the movement of the fluorescent object which is thesubject of measurement is slow. Thus, the more the attenuation curve ofthe autocorrelation function G(τ) is shifted to the right, the greaterthe size of the fluorescent object which is the subject of measurementhas become.

As shown in FIG. 14, the attenuation curve of the solution after mixingof Example V-1 (“substance-encapsulating vesicle of Example 1” in thegraph) is shifted more to the right compared to the attenuation curve ofthe QD solution (“encapsulation-target substance (QD)” in the graph)which is a encapsulation-target substance, indicating that theattenuation has become slow. Also, comparison with the FCS datadescribed in Non-patent document 2 reveals that the attenuation curve ofthe solution after mixing of Example V-1 indicates attenuation similarto the vesicle with a particle size of about 100 nm.

From the above results, it can be seen that in the solution after mixingof Example V-1, substance-encapsulating vesicles wherein QD isencapsulated in the vacant vesicles have been formed.

Comparative Example V-1: Simultaneous Mixing of a Polymer and QD

Each of the first polymer and the second polymer similar to Example V-1was dissolved in a 10 mM phosphate buffer (pH 7.4) (an aqueous medium)to a polymer concentration of 2.0 mg/ml. To the first polymer solutionobtained, QD similar to that of Example V-1 was added as anencapsulation-target substance to a concentration of 2.3×10¹⁵/ml, andthen the second polymer solution was placed in an Eppendorf tube so thatthe charge ratio becomes equal (i.e. the C/A ratio=1.0), then agitatedand mixed with a vortex mixer at 3300 rpm for 2 minutes, and thenallowed to stand for over 3 minutes. The solution obtained (solutionafter mixing) was not transparent but cloudy unlike the solution aftermixing of Example V-1.

The solution after mixing was measured by the dynamic light scatteringmethod to determine particle size distribution. The graph of particlesize distribution is shown in FIG. 15. As can be seen from FIG. 15, twoparticle size peaks were noted in stead of one particle size peak.

From the above results, it can be seen that in the solution after mixing(the solution obtained by simultaneously mixing the first polymer andthe second polymer together with QD) obtained in Comparative ExampleV-1, the polymers do not form uniform vesicles unlike Example V-1 andQD-encapsulating vesicles have not been obtained as a uniform product,either.

Example V-2: In Vivo Evaluation of a QD-Encapsulating Vesicle

Tumor model mice obtained by subcutaneously tranplanting Colon26(1.0×106/50 μl) to a hind limb of Balb/c nude mice were used on day 13after transplantation. 31.4 μl of a QD-encapsulating vesicle solutionobtained in a procedure similar to Example V-1 was diluted in PBS to atotal volume of 200 μl (QD concentration: 36 μg/μ1), and intravenouslyadministered to the tumor model mice. 1, 12, 24 and 96 hours after theadministration, fluorescent images were photographed using theIVIS(trademark) Fluorescence Imaging System (manufactured by Caliper).

For comparison, a PBS solution of QD alone supporting no vesicles (QDconcentration: 36 μg/μ1, total volume: 200 μl) was intravenouslyinjected to tumor model mice similarly to the above, and fluorescentimages were similarly photographed. For tumor model mice that did notreceive QD, fluorescent images were similarly photographed.

FIG. 16A is a fluorescent image of tumor model mice that did not receiveQD, FIG. 16B is a fluorescent image (from the top to the bottom, 1, 12,24, and 96 hours after administration) of tumor model mice that receivedQD alone, FIG. 16C is a fluorescent image of tumor model mice that thereceived QD-encapsulating vesicles (from the top to the bottom, 1, 12,24, and 96 hours after administration). In the tumor model mice thatreceived QD alone (see FIG. 16B), the blood level of QD has alreadydecreased at 12 hours after the administration, and has been virtuallyeliminated at 24 hours after the administration, whereas in the tumormodel mice that received the QD-encapsulating vesicles (see FIG. 16C),QD remains throughout the body even at 96 hours after theadministration, and specifically accumulated at high concentration inthe vicinity of the tumor. From this, it can be seen that theQD-encapsulating vesicle obtained in Example V-1 is highly excellent inretention in the blood and accumulation in the tumor. Based on thisexperiment, it is obvious that the substance-encapsulating vesicle ofthe present invention can be used very effectively as a DDS for drugs,etc.

Example Group VI: Production and Evaluation of a Positively ChargedSubstance-Encapsulating Vesicle Example VI-1: Production of a Lysozyme(Positively Charged Substance)-Encapsulating Vesicle

A procedure similar to that of Example V-1 was followed, except that QDwas replaced with lysozyme (lysozyme from chicken egg white,manufactured by Sigma, mean particle size 3 nm, molecular weight 14,000,isoelectric point (pI)=about 11) as an encapsulation-target substanceand the encapsulation-target substance in the subject solution to bemixed was added to a concentration of 1 mg/ml. The solution obtainedafter mixing was transparent.

The solution after mixing was added to a solution (in a 10 mM phosphatebuffer (0 mM NaCl), pH 7.4) containing 10 equivalents of EDC relative tothe carboxyl group contained in PEG-P(Asp) in the vesicle, and thenallowed to stand at room temperature for 12 hours to crosslink thepolymer. Lysozyme remaining in the free state in the solution withoutbeing encapsulated was removed by centrifugal ultrafiltration (VIVASPIN20 manufactured by Sartorius Sterium Biotech, a molecular weight cutoffof 300,000 was used; 300 rpm, 4° C.) to purify the solution, which wasthen measured by the dynamic light scattering method to determineparticle size distribution, mean particle size, and polydispersity index(PDI). The graph of particle size distribution is shown in FIG. 17. At amean particle size of 110 nm, the formation of monodispersion particleswas noted. The PDI was 0.087.

Subsequently, the solution after purification was measured by thefluorescence correlation spectroscopy (FCS) method to determine anautocorrelation function G(τ). For the solution of lysozyme (1 mg/ml)which is an encapsulation-target substance as well, measurement by FCSwas conducted to determine an autocorrelation function G(τ). Thetemporal attenuation curve of the autocorrelation function G(τ) obtainedis shown in FIG. 18. As shown in FIG. 18, the attenuation curve of thesolution after mixing of Example 2 (“the substance-encapsulating vesicleof Example VI-1” in the graph) is shifted more to the right compared tothe attenuation curve of lysozyme (“encapsulation-target substance(lysozyme)” in the graph) which is an encapsulation-target substance,indicating that the attenuation has become slow. Also, comparison withthe FCS data described in Non-patent document 2 reveals that theattenuation curve of the solution after mixing of Example VI-1 isshowing attenuation similar to the vesicle with a particle size of about100 nm.

From the above results, it can be seen that in the solution after mixingof Example VI-1, substance-encapsulating vesicles wherein lysozyme isencapsulated in the vacant vesicles have been formed.

Comparative Example VI-1: Simultaneous Mixing of a Polymer and Lysozyme

Each of the first polymer and the second polymer similar to Example VI-1was dissolved in a 10 mM phosphate buffer (pH 7.4) (an aqueous medium)to a polymer concentration of 2.0 mg/ml. To the first polymer solutionobtained, lysozyme similar to that of Example VI-1 was added as anencapsulation-target substance to a concentration of 1.0 mg/ml, and thenthe second polymer solution was placed in an Eppendorf tube so that thecharge ratio becomes equal (i.e. the C/A ratio=1.0), then agitated andmixed with a vortex mixer at 3300 rpm for 2 minutes, and then allowed tostand for over 3 minutes. The solution obtained (solution after mixing)was not transparent but cloudy unlike the solution after mixing ofExample V-I1.

The solution after mixing was measured by the dynamic light scatteringmethod to determine particle size distribution. The graph of particlesize distribution is shown in FIG. 19. As can be seen from FIG. 19, twoparticle size peaks were noted in stead of one particle size peak.

From the above results, it can be seen that in the solution after mixing(the solution obtained by simultaneously mixing the first polymer andthe second polymer together with lysozyme) obtained in ComparativeExample VI-1, the polymers do not form uniform vesicles unlike ExampleVI-1, and lysozyme-encapsulating vesicles have not been obtained as auniform product, either.

Example VII: Production of a Macromolecule (Cy3-Labelled PICMicelle)-Encapsulating Vesicle

A procedure similar to Example V-1 was followed except that acrosslinked fluorescence-labeled micelle (Cy3-labelled PIC micelle,Cy3-labelled PEG-P(Asp) and PEG-P(Asp-AP) (for all of them, themolecular weight of PEG is 2,000, the degree of polymerization of ionchain is 75) were mixed and prepared so as to balance the electriccharge, and then crosslinked with 10 equivalents of EDC, and similarlypurified with a molecular weight cutoff of 100,000; mean particle size38.0 nm, PDI 0.032) was used in stead of QD as an encapsulation-targetsubstance, and added to a concentration of the encapsulation-targetsubstance in the subject solution to be mixed of 1 mg/ml. The solutionobtained after mixing was transparent.

The solution after mixing was added to a solution (in a 10 mM phosphatebuffer (0 mM NaCl), pH 7.4) containing 10 equivalents of EDC relative tothe carboxyl group contained in PEG-P(Asp) in the vesicle, and thenallowed to stand at room temperature for 12 hours. The crosslinkedmicelle remaining in the free state in the solution without beingencapsulated in the vesicle was removed by centrifugal ultrafiltration(VIVASPIN 20 manufactured by Sartorius Sterium Biotech, a molecularweight cutoff of 300,000 was used; 300 rpm, 4° C.) to purify thesolution, which was then measured by the dynamic light scattering methodto determine particle size distribution, mean particle size, andpolydispersity index (PDI). At a mean particle size of 113 nm, theformation of monodispersion particles was noted. The PDI was 0.063.

Also, the solution after purification was measured by the fluorescencecorrelation spectroscopy (FCS) method to determine an autocorrelationfunction G(τ). For the solution of fluorescence-labeled micelle (1mg/ml) which is an encapsulation-target substance as well, measurementby FCS was conducted to determine an autocorrelation function G(τ). Thetemporal attenuation curve of the autocorrelation function G(τ) obtainedis shown in FIG. 20. As shown in FIG. 20, the attenuation curve of thesolution after mixing of Example VII (“the substance-encapsulatingvesicle of Example 2” in the graph) is shifted more to the rightcompared to the attenuation curve of the fluorescence-labeled micelle(“the encapsulation-target substance (Cy3-labelled PIC micelle)” in thegraph) which is an encapsulation-target substance, indicating that theattenuation has become slow. Also, comparison with the FCS datadescribed in Non-patent document 2 reveals that the attenuation curve ofthe solution after mixing of Example VII is showing attenuation similarto the vesicle with a particle size of about 100 nm.

Also, the solution after mixing was examined by a phase-contrastcryo-transmission electron microscope. A sample solution was developedonto a microgrid, excess water was blotted off with a filter paper, andthen was quick-frozen in a liquified ethane using EM CPC cryo-station(manufactured by Leica Microsystems) to prepare a specimen forexamination. Images were photographed at −170° C. using a transmissionelectron microscope JEM2011 (manufactured by JEOL Ltd.) equipped with aZernike phase plate. The electron micrographs obtained are shown inFIGS. 21A and 21B. As can be seen from FIGS. 21A and 21B, sphericalvesicle particles with a membrane thickness of about 10 nm wereobserved, confirming that they had a structure similar to the vacantvesicle.

The above results reveal that in the solution after mixing of ExampleVII, the substance-encapsulating vesicles encapsulatingfluorescence-labeled micelle in the vacant vesicles have been formed.

Example VIII: Production and Evaluation of an Enzyme(β-Glucosidase)-Encapsulating Vesicle

A procedure similar to Example V-1 was followed except that QD wasreplaced with β-glucosidase (derived from almond, manufactured by Sigma,molecular weight 10,000, isoelectric point (pI)=about 43) and theencapsulation-target substance in the subject solution to be mixed wasadded to a concentration of 1 mg/ml. The solution obtained after mixingwas transparent.

The solution after mixing was added to a solution (in a 10 mM phosphatebuffer (0 mM NaCl), pH 7.4) containing 10 equivalents of EDC relative tothe carboxyl group contained in PEG-P(Asp) in the vesicle, and thenallowed to stand at room temperature for 12 hours to crosslink thepolymer. β-glucosidase remaining in the free state in the solutionwithout being encapsulated in the vesicle was removed by centrifugalultrafiltration (VIVASPIN 20 manufactured by Sartorius Sterium Biotech,a molecular weight cutoff of 300,000 was used; 300 rpm, 4° C.) to purifythe solution, which was substituted to PBS to obtain aβ-glucosidase-encapsulating vesicle solution.

100 μl of the β-glucosidase-encapsulating vesicle solution obtained wasincubated at 37° C. for 15 minutes. Subsequently, to this solution, 100μl of 30.1 mg/ml solution of o-nitrophenyl-β-D-glucopyranoside in PBS(the abbreviated name ONPGlc, molecular weight 301) was added, andfurther incubated at 37° C. for 15 minutes. Then 50 μl of 1 mg/ml Na₂CO₃solution at a concentration of 22.1 mg/ml was added, and absorbance ataround 420 nm was determined.

ONPGlc with a molecular weight of 301 can penetrate and enter into theEDC-crosslinked vesicle. ONPGlc is a substrate for β-glucosidase, andcan produce o-nitrophenol when hydrolyzed by β-glucosidase. Sinceo-nitrophenol has a UV absorption peak at around 420 nm in which ONPGlchas no such peak, the enzyme activity of β-glucosidase can be determinedby observing this peak.

Changes with time of absorbance obtained are shown in FIG. 22. It can beseen from FIG. 22 that though the vesicle was crosslinked with EDC,β-glucosidase in the vesicle exhibited the enzyme activity without beinginactivated. It is clear from this experiment that thesubstance-encapsulating vesicle of the present invention can be usedhighly effectively as an enzyme carrier, too.

[Reference Experiment: Disturbance of Vacant Vesicle Structure byMixing]

The vacant vesicle solution obtained by a procedure similar to ExampleV-1 was added to a solution (in a 10 mM phosphate buffer (0 mM NaCl), pH7.4) containing 10 equivalents of EDC relative to the carboxyl groupcontained in PEG-P(Asp) in the vesicle, and then allowed to stand atroom temperature for 12 hours to crosslink the polymer. Aftercrosslinking, molecular weight distribution was determined by gelpermeation chromatography (GPC). For GPC, the eluent used was 10 mM PB(pH 7.4, 150 mM NaCl) and the column used was Superose 6TM 10/300 GL(manufactured by GE Healthcare), and the eluent was passed through thecolumn at a flow rate of 0.5 ml/min and detected by a UV detector (220nm) and a fluorescence detector (Ex/Em=520/550 nm).

To the vacant vesicle solution obtained by a procedure similar toExample V-1, a solution (in a 10 mM phosphate buffer (0 mM NaCl), pH7.4) containing 10 equivalents of EDC relative to the carboxyl groupcontained in PEG-P(Asp) in the vesicle was added while agitating andmixing with a vortex mixer at about 2000 rpm to conduct crosslinking.After crosslinking, molecular weight distribution was determined GPC.

GPC profiles obtained for the crosslinked solution before mixing and thecrosslinked solution after mixing are shown FIGS. 23A and 23B,respectively. In the GPC profile (see FIG. 23A) of the crosslinkedsolution before mixing, a peak appearing at about 12-13 minutesindicates the vesicle of a large molecular weight, and a peak appearingat about 30 minutes indicates a non-aggregate (an assembly of polymersnot forming vesicles) of a smaller molecular weight. On the other hand,in the GPC profile (see FIG. 23A) of the crosslinked solution aftermixing, the peak of the vesicle disappeared and the peak of thenon-aggregate is enhanced. The result shows that mixing disturbed thestructure of the vacant vesicle and broke down to small aggregates.

INDUSTRIAL APPLICABILITY

In accordance with the present invention, a vesicle(substance-encapsulating vesicle) containing/supporting a substance in acavity of a vacant vesicle obtained by polymer self-assembly in watercan be simply and effectively produced using the reproducing ability ofthe vesicle, and thus has a very high usefulness in the fields of DDSfor delivering drugs, biomaterials/function materials and the like.Specifically, the present invention has a great significance in that itdeveloped a method for encapsulating a substance whose directencapsulation into the electrostatically interacting vesicle (PICsome)is difficult, thereby expanding the application range of theencapsulation-target substance. In fact, it is remarkably useful in thatit expanded the application range of PICsome with a mean particle sizeof 100-200 nm that exhibits excellent blood retention and tumoraccumulation.

Also, in accordance with the present invention, a vesicle retainingnucleic acid in the membrane thereof is also provided, and theintra-membrane nucleic acid-containing vesicle is useful as a DDS fordelivering such nucleic acid and as biomaterials/functionally materialscomprising nucleic acid as an active ingredient. The present inventionalso permits the encapsulation of another drug in a cavity of thevesicle, and therefore is also useful as a DDS that delivers nucleicacid in combination with another drug.

1. A vesicle comprising a membrane containing: a block copolymer havingan uncharged hydrophilic segment and a cationic segment; and a nucleicacid; said membrane defining a cavity surrounded thereby.
 2. A vesicleaccording to claim 1, wherein the membrane has a trilaminar structurecomprising an outer layer, an intermediate layer, and an inner layer. 3.A vesicle according to claim 1, which has an N⁺/P ratio of higher than1.0 and lower than 3.0, the N⁺/P ratio being a mole ratio of cationicgroups of the cationic segment to phosphate groups of the nucleic acid.4. A vesicle according to claim 1, wherein the uncharged hydrophilicsegment is polyalkylene glycol.
 5. A vesicle according to claim 1,wherein the cationic segment is polyamine.
 6. A vesicle according toclaim 1, wherein the membrane further comprises a cross-linker.
 7. Avesicle according to claim 6, which has a CL/N ratio of 0.1 or higher,the CL/N ratio being a mole ratio of the cross-linker to cationic groupsof the cationic segment.
 8. A vesicle according to claim 6, wherein thecross-linker is glutaraldehyde.
 9. A vesicle according to claim 1, foruse in a drug delivery system.
 10. A drug delivery system comprising avesicle having a membrane containing: a block copolymer having anuncharged hydrophilic segment and a cationic segment, said membranedefining a cavity surrounded thereby; and a charged substanceencapsulated in the cavity.